Methods for treating brain metastasis

ABSTRACT

The present invention relates to methods for treating brain metastasis by inhibiting gap junction functionality. It is based, at least in part, on the discovery that cancer cells expressing Protocadherin 7 and Connexin 43 form gap junctions with astrocytes that promote the growth of brain metastases, and that inhibition of Protocadherin 7 and/or Connexin 43 expression in cancer cells reduce progression of brain metastases. It is further based on the discovery that treatment with gap junction inhibitors tonabersat and meclofenamate inhibited progression of brain metastatic lesions and enhanced the anti-cancer activity of the conventional chemotherapeutic agent, carboplatin.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.15/462,253, filed Mar. 17, 2017, which is a continuation ofInternational Patent Application No. PCT/US2015/051057, filed Sep. 18,2015, which claims priority to U.S. Provisional Application No.62/052,966, filed Sep. 19, 2014, to each of which priority is claimedand the contents of which are hereby incorporated by reference in theirentireties.

GRANT INFORMATION

This invention was made with government support under CA129243, CA163167and CA008748 awarded by the National Institutes of Health andW81XWH-12-0074 awarded by the Department of Defense (DoD). Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted via EFS on Sep. 13, 2019. Pursuant to 37 C.F.R. § 1.52(e)(5),the Sequence Listing text file, identified as 072734_0933SEQLIST.txt, is4,064 bytes in size and was created on Sep. 13, 2019. The SequenceListing, electronically filed on Sep. 13, 2019, does not extend beyondthe scope of the specification and thus does not contain new matter.

1. INTRODUCTION

This present invention relates to gap junction inhibitors for use intreating brain metastasis. As such, these inhibitors may be used inmethods of treating cancer patients.

2. BACKGROUND OF THE INVENTION

Brain metastases occur in 20-40% of advanced stage cancers and representthe most prevalent intracranial malignancy in adults (Gavrilovic andPosner, 2005; Maher et al., 2009). Lung and breast cancers are the mostcommon sources. Despite treatment advances at other metastatic sites,current clinical management of brain metastases affords limited diseasecontrol and most patients succumb to tumor progression less than twelvemonths after diagnosis (Gavrilovic and Posner, 2005; Stelzer, 2013). Themechanisms underlying this disease process must therefore be understoodso that they may be parlayed into rational therapeutic strategies.

The brain's unique microenvironment poses a formidable barrier tometastatic cancer cells. Recent progress has begun to unravel thecomplex cellular and molecular interactions responsible for theinitiation of brain metastases. Circulating cancer cells thatmechanically lodge in brain capillaries must first traverse thereinforced vessel walls that constitute the blood-brain barrier (BBB)(Eichler et al., 2011). Genes have been identified that mediate cancercell extravasation through the BBB in experimental models and predictbrain metastasis in the clinic (Bos et al., 2009; Li et al., 2013). Onceinside the brain parenchyma, metastatic cells remain associated with themicrovasculature (Kienast et al., 2010; Lorger and Felding-Habermann,2010). Expression of the cell adhesion molecule LCAM in the cancer cellsmediates their tight adhesion to the abluminal capillary basal lamina asa requirement for the initiation of metastatic outgrowth (Valiente etal., 2014). Wnt is one of the signaling pathways supporting theoutgrowth (Nguyen et al., 2009). However, the vast majority of cancercells that infiltrate the brain perish (Chambers et al., 2002; Heyn etal., 2006; Kienast et al., 2010), and they are rejected by the mostabundant cell type in the brain, the astrocyte (Valiente et al., 2014).

Functionally pleiotropic, astrocytes maintain the BBB, orchestrateneurovascular coupling, sustain homeostasis of a tissue under stringentmetabolic demands (Oberheim et al., 2012) and react acutely againstdisturbances like injury or infiltrating cells (Pekny and Nilsson,2005). Reactive astrocytes generate plasmin, which mobilizes thepro-apoptotic cytokine FasL to kill infiltrating cancer cells (Valienteet al., 2014). Plasmin additionally cleaves cell surface LCAM in thecancer cells to suppress their ability to coopt the vasculature(Valiente et al., 2014). To evade astrocyte attack, brain metastaticcells from breast cancer and lung cancer express serpin inhibitors ofplasminogen activator (PA) (Valiente et al., 2014). Although theseobservations indicate that astrocytes guard the brain against metastaticinvasion, there is also evidence that the role of astrocytes inmetastasis may not be uniformly antagonistic. In vitro, astrocyteco-culture protects melanoma cell lines from chemotherapeutic drugs (Kimet al., 2011), and in vivo astrocytes can activate Notch signaling incancer cells (Xing et al., 2013).

3. SUMMARY OF THE INVENTION

The present invention relates to methods for treating brain metastasisby inhibiting gap junction functionality. It is based, at least in part,on the discovery that cancer cells expressing Protocadherin 7 andConnexin 43 form gap junctions with astrocytes that promote the growthof brain metastases, and that inhibition of Protocadherin 7 and/orConnexin 43 expression in cancer cells reduces progression of brainmetastases. It is further based on the discovery that treatment with gapjunction inhibitors tonabersat and meclofenamate inhibited progressionof brain metastatic lesions and enhanced the anti-cancer activity of theconventional chemotherapeutic agent, carboplatin.

Certain non-limiting embodiments provide for a method for treating asubject having a cancer comprising administering, to the subject, anamount of a gap junction inhibitor that inhibits metastatic progressionof the cancer in the brain. In particular non-limiting examples, the gapjunction inhibitor is a Connexin 43 inhibitor or a Protocadherin 7inhibitor, or a combination thereof. In particular non-limitingexamples, the inhibitor is tonabersat or meclofenamate or a combinationthereof. In particular non-limiting examples, the cancer is breastcancer or lung cancer, and/or the cancer cells of the subject expressConnexin 43 and/or Protocadherin 7. In particular non-limiting examples,the method further comprises administering, to the subject, atherapeutically effective amount of an anti-cancer agent such as, butnot limited to, carboplatin. When the method of the invention isapplied, the subject may be known to have one or more brain metastases,or alternatively, was not known to have a brain metastasis prior totreatment.

Certain non-limiting embodiments provide for a method for inhibitinggrowth and/or survival of metastatic cancer cells in the brain of asubject, comprising treating the subject with a therapeuticallyeffective amount of a gap junction inhibitor. In particular non-limitingexamples, the gap junction inhibitor is a Connexin 43 inhibitor or aProtocadherin 7 inhibitor, or a combination thereof. In particularnon-limiting examples, the inhibitor is tonabersat or meclofenamate or acombination thereof. In particular non-limiting examples, the cancer isbreast cancer or lung cancer, and/or the cancer cells of the subjectexpress Connexin 43 and/or Protocadherin 7. In particular non-limitingexamples, the method further comprises administering, to the subject, atherapeutically effective amount of an anti-cancer agent such as, butnot limited to, carboplatin. When the method of the invention isapplied, the subject may be known to have one or more brain metastases,or alternatively, was not known to have a brain metastasis prior totreatment.

Certain non-limiting embodiments provide for a method for treating brainmetastasis in a subject having a cancer, comprising administering, tothe subject, a therapeutically effective amount of a gap junctioninhibitor. In particular non-limiting examples, the gap junctioninhibitor is a Connexin 43 inhibitor or a Protocadherin 7 inhibitor, ora combination thereof. In particular non-limiting examples, theinhibitor is tonabersat or meclofenamate or a combination thereof. Inparticular non-limiting examples, the cancer is breast cancer or lungcancer, and/or the cancer cells of the subject express Connexin 43and/or Protocadherin 7. In particular non-limiting examples, the methodfurther comprises administering, to the subject, a therapeuticallyeffective amount of an anti-cancer agent such as, but not limited to,carboplatin. When the method of the invention is applied, the subjectmay be known to have one or more brain metastases, or alternatively, wasnot known to have a brain metastasis prior to treatment.

Certain non-limiting embodiments provide for, in a subject having acancer, a method of preventing metastasis of the cancer to the brain,comprising administering, to the subject, a therapeutically effectiveamount of a gap junction inhibitor. In particular non-limiting examples,the gap junction inhibitor is a Connexin 43 inhibitor or a Protocadherin7 inhibitor, or a combination thereof. In particular non-limitingexamples, the inhibitor is tonabersat or meclofenamate or a combinationthereof. In particular non-limiting examples, the cancer is breastcancer or lung cancer, and/or the cancer cells of the subject expressConnexin 43 and/or Protocadherin 7. In particular non-limiting examples,the method further comprises administering, to the subject, atherapeutically effective amount of an anti-cancer agent such as, butnot limited to, carboplatin. When the method of the invention isapplied, the subject may be known to have one or more brain metastases,or alternatively, was not known to have a brain metastasis prior totreatment.

Certain non-limiting embodiments provide for in a subject having acancer, a method of reducing the risk of detectable metastasis of thecancer to the brain, comprising administering, to the subject, atherapeutically effective amount of a gap junction inhibitor. Inparticular non-limiting examples, the gap junction inhibitor is aConnexin 43 inhibitor or a Protocadherin 7 inhibitor, or a combinationthereof. In particular non-limiting examples, the inhibitor istonabersat or meclofenamate or a combination thereof. In particularnon-limiting examples, the cancer is breast cancer or lung cancer,and/or the cancer cells of the subject express Connexin 43 and/orProtocadherin 7. In particular non-limiting examples, the method furthercomprises administering, to the subject, a therapeutically effectiveamount of an anti-cancer agent that can attain therapeutic levels in thebrain, such as, but not limited to, carboplatin. When the method of theinvention is applied, the subject may be known to have one or more brainmetastases, or alternatively, was not known to have a brain metastasisprior to treatment.

Certain non-limiting embodiments provide for, in a subject having acancer, a method of reducing the risk of detectable metastasis of thecancer to the brain, comprising administering, to the subject, atherapeutically effective amount of a Protocadherin 7 inhibitor. Inparticular non-limiting examples, the Protocadherin 7 inhibitor is aninterfering RNA. In particular non-limiting examples, the cancer isbreast cancer or lung cancer, and/or the cancer cells of the subjectexpress Connexin 43 and/or Protocadherin 7. In particular non-limitingexamples, the method further comprises administering, to the subject, atherapeutically effective amount of an anti-cancer agent such as, butnot limited to, carboplatin. When the method of the invention isapplied, the subject may be known to have one or more brain metastases,or alternatively, was not known to have a brain metastasis prior totreatment.

Certain non-limiting embodiments provide for a method for lengtheningthe period of survival of a subject having a cancer, comprisingadministering to the subject an effective amount of a gap junctioninhibitor, for example, wherein administering the gap junction inhibitorinhibits metastatic progression of the cancer in the brain. Inparticular non-limiting examples, the gap junction inhibitor is aConnexin 43 inhibitor or a Protocadherin 7 inhibitor, or a combinationthereof. In particular non-limiting examples, the inhibitor istonabersat or meclofenamate or a combination thereof. In particularnon-limiting examples, the cancer is breast cancer or lung cancer,and/or the cancer cells of the subject express Connexin 43 and/orProtocadherin 7. In particular non-limiting examples, the method furthercomprises administering, to the subject, a therapeutically effectiveamount of an anti-cancer agent such as, but not limited to, carboplatin.When the method of the invention is applied, the subject may be known tohave one or more brain metastases, or alternatively, was not known tohave a brain metastasis prior to treatment.

Certain non-limiting embodiments provide for an assay for evaluating gapjunction activity, for example assessing inhibition, by measuring levelsof cGAMP, where a decrease in cGAMP correlates with gap junctioninhibition. Particular non-limiting embodiments provide for a method forinhibiting growth and/or survival of metastatic cancer cells in thebrain of a subject, comprising treating the subject with atherapeutically effective amount of a gap junction inhibitor thatproduces a decrease in cGAMP relative to the level of cGAMP in theabsence of that amount of gap junction inhibitor. Further non-limitingembodiments provide for a method of determining whether a brain tumor ormetastatic brain tumor in a subject will receive therapeutic benefitfrom treatment with a gap junction inhibitor, comprising determiningwhether, in a sample from said tumor, exposure to a gap junctioninhibitor leads to a decrease in cGAMP, where a decrease in cGAMP isindicative of therapeutic benefit.

4. BRIEF DESCRIPTION OF FIGURES

FIGS. 1A-1K provide Cx43 and PCDH7 association with brain metastasis.(FIG. 1A) GFP+H2030-BrM3 cells (green) are surrounded by GFAP+ activatedastrocytes (red) in the brain parenchyma at early (day 7) and later (day21) time points following intracardiac inoculation in mice. Blue,collagen IV (ColIV) staining in vessels. Scale bar, 10 μm. (FIG. 1B)Cx43 staining (arrowhead) at the interface of GFP+H2030-BrM3 (green) andGFAP+ astrocytes (blue). Scale bar, 10 μm. (FIG. 1C) Representativeimages of Cx43 staining in human brain metastasis samples fromtriple-negative breast cancer and non-small cell lung carcinoma. Theproportion of CX43-positive samples was quantified in primary (Iry)tumours, brain metastases, and normal lung tissues. Scale bar, 100 μm.(FIG. 1D) Representative images and quantification of Cx43immunostaining in matched primary and brain metastatic samples fromnon-small lung carcinoma patients. Scale bar, 100 μm. (FIG. 1E)Schematic illustration of dye transfer assay. (FIG. 1F) Quantificationof dye transfer from astrocytes to cancer cells. Histograms show redfluorescent signal in parental (Par) and BrM cells. All values aremean±S.E.M. (n=3 biological replicates). n=3 independent experiments.(FIG. 1G-1I) Cx43 and PCDH7 western immunoblotting in the indicatedparental and brain metastatic derivatives ((FIG. 1G) n=3 independentexperiments), in brain metastatic cells compared to brain cell types((FIG. 1H) n=2 independent experiments), and in MDA231 derivativesmetastatic to brain, lung (LM) or bone (BoM) ((FIG. 1I) n=2 independentexperiments). (FIGS. 1J-1K) Kaplan-Meier plot of cumulative brainmetastasis-free survival in 189 cases of triple-negative breast cancer(FIG. 1J) and 129 cases (MSKCC set2) and 58 cases (GSE3141) of lungadenocarcinoma (FIG. 1K), based on Cx43/PCDH7 expression in the primarytumour.

FIGS. 2A-2G provide Cx43/PCDH7 carcinoma-astrocyte gap junctions mediatebrain metastasis. (FIG. 2A) Histograms (top) and quantification (bottom)of dye transfer from astrocytes to control and Cx43-depleted orPCDH7-depleted brain metastatic cells. Values are mean±S.E.M. (n=3biological replicates). n=3 independent experiments. (FIG. 2B)Luciferase complementation assay to detect Cx43-PCDH7 interactions. NLucand CLuc, N-terminal and C-terminal firefly luciferase halves. The table(top) numerically identifies the cell line combinations used in theassays (bottom), and bioluminescence imaging (BLI) of a representativeplate. BLI (FIG. 2C) and quantification (FIG. 2D) of brain metastaticlesions formed by control, Cx43-depleted, or PCDH7-depleted brainmetastatic cells. n=3 independent experiments. (FIGS. 2E, 2F) Wild type(WT) or T154A mutant (Mut) Cx43 was re-expressed in Cx43-depletedMDA231-BrM2 cells (Cx43 sh2). The cells were subjected to astrocyte dyetransfer analysis ((FIG. 2E) n=3 independent experiments), or to brainmetastasis assays and BLI quantification ((FIG. 2F) n=2 independentexperiments). (FIG. 2G) Schematic summary of Cx43- and PCDH7-mediatedinteractions between cancer cells and astrocytes in brain metastasis.

FIGS. 3A-3I provide gap junctions activate STAT1 and NF-κB pathways incancer cells. (FIG. 3A) Signaling pathway analysis of TRAP-Seq data fromMDA231-BrM2 cells after co-culture with astrocytes. Control (Ctrl) orCx43-depleted MDA231-BrM2 cells expressing an L10a-GFP ribosomal proteinfusion were co-cultured with astrocytes for 24 h prior to polysomeimmunoprecipitation and mRNA sequencing. Heatmap depicts blue(down-regulated) and red (up-regulated) pathways. n=2 biologicalreplicates. (FIGS. 3B, 3C) STAT1 and NF-κB p65 phosphorylation inMDA231-BrM2 cells after a 2 h incubation with conditioned media (CM)from astrocyte co-culture. CM were collected after 24 h co-culture ofastrocytes with control or Cx43-depleted MDA231-BrM2 cells (FIG. 3B), orfrom Cx43-depleted MDA231-BrM2 cells that were transduced with wild typeCx43 (WT) or Cx43(T154A) mutant (Mut) (FIG. 3C). n>3 independentexperiments. (FIG. 3D) ELISA of IFNα and TNFα in CM from astrocyteco-cultures with the indicated MDA231-BrM2 cells. All values aremean±S.E.M. (n=4 technical replicates). n>2 independent experiments.(FIG. 3E) Relative mRNA levels of IFNA and TNFA in astrocytesre-isolated after co-culture with MDA231-BrM2 cancer cells. All valuesare mean±S.E.M. (n=3 biological replicates). n=2 independentexperiments. (FIG. 3F) Relative levels of cleaved caspase 3 inMDA231-BrM2 cells treated with various concentrations of carboplatin(Carbo) in the presence or absence of 10 units/ml (39 units/ng) IFNαA or10 μg/ml TNFα. All values are mean±S.E.M. (n=5 technical replicates).n=3 independent experiments. (FIG. 3G) STAT1 levels in control andSTAT1-knockdown MDA231-BrM2 cells. (FIG. 3H) NF-κB renilla luciferasereporter assay in MDA231-BrM cells expressing control pBABE or SR-IκBαvector. All values are mean±S.E.M. (n=3 technical replicates). (FIG. 3I)Quantification of BLI signal from brain metastases formed by control,STAT1-knockdown, and SR-IκBα MDA231-BrM2 cells. n=2 independentexperiments.

FIGS. 4A-4H provide gap junctions mediate a cytosolic dsDNA response inastrocytes. (FIG. 4A) MDA231-BrM2 cells expressing control shRNA (Ctrlsh) or shRNA targeting Cx43, were cultured for 18 h with or withoutastrocytes, and subjected to immunoblotting analysis of phosphorylatedTBK1 and IRF3 (n=3 independent experiments). (FIG. 4B) MDA231-BrM2alone, astrocytes alone, or 18 h co-cultures, were harvested for samplepreparation and cGAMP analysis by LC-MS/MS. Histogram (right)corresponds to normalized cGAMP peaks in (left), and is representativeof 5 biological replicates. n=3 independent experiments. See also FIG.16. (FIG. 4C) Representative images of dual immunofluorescent stainingof IRF3 and GFP. DAPI, nuclear staining. In co-cultures: white arrows,nuclear accumulation of IRF3 in astrocytes; green arrows, evendistribution of IRF3 in GFP+ MDA231-BrM2 cells. Scale bar, 20 μm. n=2independent experiments. (FIG. 4D) Quantification of dsDNA in theindicated cellular fractions from 2×10⁷ cells. Values are mean±S.E.M.(n=3 biological replicates). n=2 independent experiments. (FIG. 4E)Representative image of immunofluorescence staining of dsDNA, GFP, andCox IV (mitochondrial marker) in MDA231-BrM2 cells. DAPI, nuclearstaining. Scale bar, 10 μm. n=2 independent experiments. (FIGS. 4F, 4G)EdU labeled MDA231-BrM2 cells were co-cultured with astrocytes for 6 h.Transfer of EdU-labeled DNA from cancer cells to astrocytes wasvisualized using confocal microscopy (FIG. 4F), or quantified by flowcytometry (FIG. 4G). Cancer cells and astrocytes are delineated by greenand white dotted lines, respectively. Scale bar, 10 μm. Values aremean±S.E.M. (n=3 biological replicates, n=2 independent experiments).(FIG. 4H) Schematic summary of gap junction mediated anti-dsDNAresponse, production of IFNα and TNFα in astrocytes, and consequentactivation of STAT1 and NF-κB pathways in cancer cells to support brainmetastasis.

FIGS. 5A-5I provide inhibition of gap junction activity controls brainmetastatic outgrowth. (FIG. 5A) Dye transfer from astrocytes toMDA231-BrM2 cells in the presence of the indicated concentrations ofTonabersat or meclofenamate. n≥3 independent experiments. (FIG. 5B)ELISA of IFNα and TNFα in conditioned media from co-cultured MDA231-BrM2cell and astrocytes in the presence of Tonabersat (Tona) ormeclofenamate (Meclo) with indicated concentrations. All graphs shownare mean±S.E.M. (n=4 technical replicates). n=2 independent experiments.(FIG. 5C) Tonabersat or meclofenamate was administered daily startingone day after cancer cell inoculation in mice. Brain metastatic lesionswere quantified based on BLI. n=2 independent experiments. (FIG. 5D) GFPstaining of 14-day brain metastatic lesions. Representative images showlarge, progressive lesions. DAPI, nuclear staining. Scale Bar, 40 μm.n=10 experimental mice. (FIG. 5E) 14 days after inoculation withMDA231-BrM2 cells transduced with inducible control, CX43 or PCDH7shRNAs, mice were treated with doxycycline and carboplatin, asillustrated in the scheme. Brain metastatic lesions were quantifiedbased on BLI. (FIGS. 5F, 5G) Representative images of matched ex vivobrain BLI and red fluorescence imaging. n=2 independent experiments.(FIG. 5H) 14 days after inoculation with MDA231-BrM2 cells, mice weretreated with Tonabersat, meclofenamate, and carboplatin. Following theindicated regimens, brain metastatic lesions were quantified based onBLI. n=2 independent experiments (FIG. 51I).

FIGS. 6A-6D provide cancer cell-astrocyte interactions. (FIG. 6A) Cancercells used in this study. (FIG. 6B) Astrocyte co-culture protects cancercells. As illustrated in schema (left), cleaved caspase 3+/GFP+apoptotic BrM cells were quantified after sFasL- or chemo-treatments.n=3 independent experiments. (FIGS. 6C, 6D) Gap junction communicationsbetween astrocytes and BrM cells. Time-lapse images of dye transfer fromMDA231-BrM2 cells to astrocytes (FIG. 6C). Scale bars, 100 μm.Quantification of dye transfer from astrocytes to MDA231-BrM2 cells byflow cytometry over time (FIG. 6D). n=3 independent experiments.

FIGS. 7A-7G provide elevated expression of Cx43 and PCDH7 in brainmetastatic cancer cells and astrocytes. (FIG. 7A) Cx43 and PCDH7 mRNA inparental (Par) and BrM cells. Values are mean±S.E.M. (n=3 technicalreplicates). n=3 independent experiments. (FIG. 7B) Cx43 and PCDH7western blotting in ErbB2 parental and brain cells, as well as Kras/p53cell lines. n=3 independent experiments. (FIG. 7C) Cx43 and PCDH7 mRNAin BrM cells compared to brain cells. n=3 independent experiments. (FIG.7D) Cx26 and Cx30 mRNA in MDA231 parental (Par) and the metastaticderivatives of brain (BrM2), lung (LM) and bone (BoM). (FIG. 7E)Kaplan-Meier plot illustrates the probability of cumulative metastasisfree survival in 63 cases (GSE8893) of lung adenocarcinoma based onCx43/PCDH7 expression in the primary tumour. (FIGS. 7F, 7G) Knockdown ofCx43 and PCDH7 with short hairpin RNAs (shRNA) as assessed by RT-PCR(FIG. 7F) and western blotting (FIG. 7G). Ctrl, control. Values aremean±S.E.M. (n=3 technical replicates). n=3 independent experiments.

FIGS. 8A-8H provide PCDH7 facilitates gap junction communication. (FIGS.8A, 8B) Histograms and quantification of dye transfer from astrocytes tocontrol and Cx43-depleted or PCDH7-depleted Kras/p53-393N1 cells (FIG.8A), and from astrocytes to control or Cx43-depleted MDA231-BrM2 cells,in comparison to Carbenoxolone (50 uM) treatment (FIG. 8B). (FIGS. 8C,8D) PCDH7 in astrocytes facilitate gap junctions. PCDH7 western blottingin control or PCDH7-depleted astrocytes (FIG. 8C). Quantification of dyetransfer from MDA231-BrM2 cells to PCDH7-depleted astrocytes (FIG. 8D).(FIG. 8E) Quantification of dye transfer from human brain microvascularendothelial cells (HBMEC) to control, Cx43- or PCDH7-depletedMDA231-BrM2 cells. (FIG. 8F) Dye transfer from MDA231-BrM2 cells to amixed population of astrocytes and HBMEC. (FIG. 8G) Quantification ofdye transfer from control or Cx43-depleted MDA231-BrM2 cells to humanmicroglia. (FIG. 8H) As illustrated in schema, Cx43 mRNA in MDA231-BrM2cells (left) or astrocytes (right) was detected after 24 h co-culture,separated by transwell, with microglia, astrocytes or cancer cells. Fordye transfer assays, values are mean±S.E.M. (n=3 biological replicates).n≥2 independent experiments. In h, values are mean±S.E.M. (n=4biological replicates).

FIGS. 9A-9E provide Cx43 directly interacts with PCDH7, but not with Ecadherin or N cadherin. (FIG. 9A) Cx43 and PCDH7 western immunoblottingin cancer cells overexpressing fusion proteins. (FIG. 9B) Quantificationof BLI after co-culture of Cx43-CLuc/PCDH7-NLuc(+) cancer cells andastrocytes for 15 min. c-e, Luciferase split assay to detect Cx43-Ecadherin or Cx43-N cadherin interactions. NLuc and CLuc: N-terminal andC-terminal firefly luciferase halves. The table (FIG. 9C) numericallyidentified the cell line combinations used in the assays, westernimmunoblotting (FIG. 9D) indicated E or N cadherin expression in cancercells overexpressing fusion proteins, and bioluminescence imaging (BLI)of a representative plate (FIG. 9E). n≥2 independent experiments.

FIGS. 10A-10E provide Inhibition of gap junction activity prevents brainmetastatic outgrowth. (FIG. 10A) Bioluminescent imaging (BLI)quantification of brain metastatic lesions formed by control (Ctrl),Cx43- or PCDH7-depleted Kras/p53-393N1 cells. n=2 independentexperiments. (FIG. 10B) Representative images of GFP+ brain metastaticlesions formed by control, Cx43- or PCDH7-depleted MDA231-BrM2 cells.Brain sections or brain metastatic lesions are delineated by dottedwhite line or dotted red line, respectively. Scale bar, 1000 μm. (FIG.10C) BLI (images) and quantification (bar graph) of lung metastaticlesions formed by MDA231-BrM2 cells. Values are mean±S.E.M. (n=5 mice ineach group). n=2 independent experiments. (FIGS. 10D, 10E) Gapjunction-mediated brain metastasis requires channel function of Cx43.Wild type (WT) or T154A mutant (Mut) Cx43 was re-expressed in Cx43depleted MDA231-BrM2 cells (CX43 sh2). Cx43 expression was detected bywestern blotting (FIG. 10D) and brain metastatsis formed by these cellswas quantified by BLI (FIG. 10E). n=2 independent experiments.

FIGS. 11A-11D provide role of Cx43 and PCDH7 in brain metastasis. (FIG.11A) Cx43 and PCDH7 do not mediate trans-BBB Migration. Quantificationof control (Ctrl), Cx43- or PCDH7-depleted MDA231-BrM2 cells in 7-daybrain lesions. Values are mean±S.E.M. (n=5 brains in each group). (FIG.11B) Cx43 and PCDH7 mediate cancer cell colonization in 14-day brainlesions. Representative images are GFP (green) and Ki67 (red) staining.DAPI, nuclear staining. Scale bar, 20 μm. Bar graph is the proportion ofKi67+ cancer cells. Values are mean±S.E.M. (n=5 brains in each group).(FIG. 11C) Cx43 and PCDH7 mediate cancer cell survival. Brain sliceassays. Representative images are GFP (green) and cleaved caspase 3(Casp3)(red) staining. Scale bar, 30 μm. Histogram is the proportion ofcaspase 3+ apoptotic cancer cells. Values are mean±S.E.M. (n=5 brainslices in each group). Scale bars, 30 μm. (FIG. 11D) Cx43 and PCDH7 donot affect vascular cooption of cancer cells in 14-day brain lesions.Representative images are GFP (green) staining and vascular structurefilled with TRITC dextran (red). Scale bar, 20 μm. n=2 independentexperiments.

FIGS. 12A-12D provide translating ribosome affinity purification (TRAP)and cytokine array. (FIG. 12A) Schematic illustration of TRAPexperimental set up to isolate translating mRNA from MDA231-BrM2 cellsunder 3 conditions (#1, #2, #3). (FIG. 12B) Principle component (PC)analysis of TRAP mRNA sequencing. (FIG. 12C) Scatter plot of log 2fold-changes regulated by astrocytes and gap junction communicationsbetween BrM cells and astrocytes. (FIG. 12D) STAT1 and NF-κB p65phosphorylation in H2030-BrM3 cells after a 2 h incubation withconditioned media (CM) from astrocyte co-cultures. CM were collectedafter 24 h co-culture of astrocytes with control or Cx43-depletedH2030-BrM3 cells. n=3 independent experiments.

FIGS. 13A-13F provide gap junction-generated signaling activates IFN andNF-Kb pathways in cancer cells. (FIG. 13A) Cytokine array analysis ofthe conditioned media collected after 24 h co-culture of astrocytes withcontrol or Cx43-depleted MDA231-BrM2 cells. Log 2 fold-changes wereplotted. (FIG. 13B) ELISA of IFNα and TNFα in CM from astrocyteco-cultures with the indicated H2030-BrM3 cells. All values shown aremean±S.E.M. (n=4 technical replicates). n=2 independent experiments.(FIG. 13C) Relative levels of cleaved caspase 3 in H2030-BrM3 cellstreated with various concentrations of Taxol in the presence or absenceof 10 units/ml (39 units/ng) recombinant IFNαA or 10 μg/ml recombinantTNFα. All values are mean±S.E.M. (n=5 technical replicates). n=3independent experiments. (FIGS. 13D, 13E) STAT1 levels in control andSTAT1-knockdown H2030-BrM3 cells. (FIG. 13F) Quantification of BLIsignal from brain metastases formed by control, STAT1-knockdown cells.n=2 independent experiments.

FIGS. 14A-14G provide gap junctions initiate cytosolic DNA response inastrocytes. (FIG. 14A) Control or Cx43-deplated H2030-BrM3 cells wereco-cultured for 18 h with or without astrocytes, and subjected toimmunoblotting analysis of phosphorylated TBK1 and IRF3 (n=3 independentexperiments). (FIG. 14B) cGAMP identification. The peak at 4.47 mincontains all 3 SRM transitions specific for cGAMP. RT: retention time,AA: automatically integrated peak area. (FIG. 14C) Quantification ofdsDNA in the indicated cellular fractions from 2×10⁷ H2030-BrM3 cells.Values are mean±S.E.M. (n=3 biological replicates). n=2 independentexperiments. (FIG. 14D) Ratio of cytosol dsDNA and nuclear dsDNA inindicated cancer cells and non-neoplastic cells. (FIG. 14E)Representative image of immunofluorescent staining of dsDNA, GFP, Cox IV(mitochondria marker) in H2030-BrM3 cells. DAPI, nuclear staining. Scalebar, 10 μm. (FIG. 14F) Representative image of immunofluorescentstaining of dsDNA, Cox IV (mitochondria marker) in astrocytes. DAPI,nuclear staining. Phalloidin, cytoskeletal staining. Scale bar, 10 μm.(FIG. 14G) EdU labeled H2030-BrM3 cells were co-cultured with astrocytesfor 6 h. Transfer of EdU-labeled DNA from cancer cells to astrocytes wasvisualized using con-focal microscopes. Cancer cells or astrocytes weredelineated by green or white dotted lines, respectively. Scale bar, 10μm. n=2 independent experiments.

FIGS. 15A-15G provide inhibition of gap junction activity prevents brainmetastatic outgrowth. (FIGS. 15A-15D) Following treatment withTonabersat (Tona) or meclofenamate (Meclo) (FIG. 15A), brain metastasis(FIG. 15B), primary tumour growth in mammary fat pads (FIG. 15C), orlung metastasis (FIG. 15D) were quantified by BLI. Values aremean±S.E.M. (n=5 mice in each group). n=2 independent experiments.(FIGS. 15E, 15F) Knockdown of Cx43 and PCDH7 in MDA231-BrM2 cells withtet-on inducible short hairpin RNAs (shRNA), as assessed by RT-PCR (FIG.15E) and Western immunoblotting (FIG. 15F), after doxycycline treatmentin vitro. n=2 independent experiments. (FIG. 15G) Brain ex vivoBioluminescent imaging (BLI) 14 days after inoculation of MDA231-BrM2cells.

FIG. 16 provides confirmation of cGAMP identification. A pooled samplefrom all experimental conditions shown in FIG. 4b analyzed by LC-MS/MS.Only the peak at 4.47 min contains all 3 SRM transitions specific forcGAMP. The peak at 4.47 min is increased by the addition of 5 μL of 40nM cyclic [G(2′,5′)pA(3′,5′)p] (cGAMP) to the pooled sample. As internaland negative control, c-di-GMP contains all 2 SRM transitions at 4.97min peak and the peak does not change by adding standard cGAMP. dRT:retention time, AA: automatically integrated peak area.

5. DETAILED DESCRIPTION

For clarity and not by way of limitation the detailed description of theinvention is divided into the following subsections:

(i) Gap junction inhibitors;

-   -   (a) Connexin 43 inhibitors; and    -   (b) Protocadherin 7 inhibitors;    -   (c) Assay for gap junction activity/inhibition;

(ii) cancer targets;

(iii) pharmaceutical formulations; and

(iv) methods of treatment.

5.1 Gap Junction Inhibitors

The present invention provides inhibitors of gap junctions (e.g., gapjunction antagonists) for use in the disclosed methods. In certainembodiments, gap junction inhibitors can include compounds, smallmolecules, chemicals, polypeptides, nucleic acids and proteins thatinhibit and/or reduce the expression and/or activity of gap junctioncomponents or inhibit and/or reduce the formation, patency, signalingand/or activity of gap junctions.

In certain non-limiting embodiments, gap junction inhibitors that aresmall molecules include carbenoxolone, glycyrrhetinic acid, quinine,quinidine, mefloquine, heptanol, octanol, anandamide, fenamates,2-aminoethoxy-diphenyl-borate (2-APB), retinoic acid, oleamide,spermine, aminosulfonates, sodium propionate, tonabersat andmeclofenamate (meclofenamic acid). Additional non-limiting examples ofgap junction inhibitors are disclosed in U.S. Pat. Nos. 5,843,989;6,211,211; 7,632,866, 6,251,931; 7,704,946; and PCT Patent ApplicationNo. WO 1999/026584.

In certain embodiments, the gap junction inhibitor comprises a compoundof Formula I having the following structure:

In certain embodiments, the gap junction inhibitor comprises a compoundof Formula II having the following structure:

In certain embodiments, the gap junction inhibitor comprises a compoundof Formula III having the following structure:

In certain non-limiting embodiments, the gap junction inhibitor can be asalt, a stereoisomer, an analog or a derivative form of the compounds ofFormulas I-III. For example, and not by way of limitation, the gapjunction inhibitor can include a sodium salt form of Formula II.

In certain non-limiting embodiments, the gap junction inhibitor can bean antibody or antibody fragment that can partially or completely blockgap junction formation and/or gap junction patency between cells, gapjunction signaling and/or activity. See, for example, ErnestoOviedo-Orta et al., The FASEB Journal, Vol. 15: 768-774 (2001). Incertain non-limiting embodiments, the gap junction inhibitor can be ananti-Connexin compound and/or a Connexin mimetic peptide. See, forexample, Evans and Boitano, Biochem. Soc. Trans., Vol. 29(4):606-612(2001); Dahl, Biophys. J., Vol. 67(5):1816-1822 (1994); European PatentApplication Nos. EP2510939 and EP2252320; and U.S. Patent ApplicationNo. 2009/0142295.

Further non-limiting examples of gap junction inhibitors includeribozymes, antisense oligonucleotides, short hairpin RNA (shRNA)molecules and siRNA molecules that specifically inhibit and/or reducethe expression or activity of gap junction components. A “ribozyme”refers to a nucleic acid capable of cleaving a specific nucleic acidsequence. In certain non-limiting embodiments, a ribozyme refers to RNAmolecules that contain anti-sense sequences for specific recognition,and an RNA-cleaving enzymatic activity, see, for example, U.S. Pat. No.6,770,633. In contrast, “antisense oligonucleotides” generally are smalloligonucleotides complementary to a part of a gene to impact expressionof that gene. Gene expression can be inhibited through hybridization ofan oligonucleotide to a specific gene or messenger RNA (mRNA) thereof.Methods for using antisense techniques for specifically inhibiting geneexpression of genes whose sequence is known are well known in the art(e.g., see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323;6,107,091; 6,046,321; and 5,981,732). “Small interfering RNA” or “shortinterfering RNA” or “siRNA” or “short hairpin RNA” or “shRNA” are formsof RNA interference (RNAi). An interfering RNA can be a double-strandedRNA or partially double-stranded RNA molecule that is complementary to atarget nucleic acid sequence. Micro RNAs (miRNA) can also fall in thiscategory. Various modifications to the oligonucleotides of the presentinvention, e.g., antisense, shRNA or siRNA molecules, can be introducedas a means of increasing intracellular stability and half-life.Non-limiting examples of such modifications include the addition offlanking sequences of ribonucleotides or deoxyribonucleotides to the 5′and/or 3′ ends of the molecule, or the use of atypical or non-naturallyoccurring residues such as phosphorothioate or 2′-O-methyl rather thanphosphodiesterase linkages within the oligonucleotide backbone.

The RNA molecules of the invention can be expressed from a vector orproduced chemically or synthetically. Methods for selecting anappropriate dsRNA or dsRNA-encoding vector are well known in the art forgenes whose sequence is known (e.g., see Tuschl, T. et al. (1999);Elbashir, S. M. et al. (2001); Hannon, G J. (2002); McManus, M T. et al.(2002); Brummelkamp, T R. et al. (2002); U.S. Pat. Nos. 6,573,099 and6,506,559; and PCT Patent Application Nos. WO 2001/036646, WO1999/032619 and WO 2001/068836).

5.1.1 Connexin 43 Inhibitors

In certain non-limiting embodiments, the gap junction inhibitor can bespecific for a gap junction component. For example, and not by way oflimitation, gap junction components include the Connexin family ofproteins. A non-limiting example of a Connexin protein is Connexin 43(Cx43), which is encoded by the gene gap junction protein, al (gial). ACx43 nucleic acid or protein may be a human Cx43 nucleic acid having thesequence as set forth in NCBI database accession no. NM_000165,NG_008308 or M65188, or a nucleic acid encoding a human Cx43 proteinmolecule that has the amino acid set forth in NCBI database accessionno. NP_000156. According to the present invention, inhibitors of theexpression and/or function of such Cx43 nucleic acids and/or proteinsmay be used as gap junction inhibitors. For example, and not by way oflimitation, a gap junction inhibitor can include a Cx43 inhibitor suchas, but not limited to, ioxynil or ioxynil octanoate. In certainembodiments, a Cx43 inhibitor can include a Cx43 antibody, antibodyfragment or a mimetic peptide (see Danesh-Meyer et al., Brain,135:506-520 (2012)).

One non-limiting example of a gap junction inhibitor comprises anantisense, shRNA or siRNA nucleic acid sequence homologous to at least aportion of a Cx43 nucleic acid sequence, disclosed above, wherein thehomology of the portion relative to the Cx43 sequence is at least about75 or at least about 80 or at least about 85 or at least about 90 or atleast about 95 or at least about 98 percent, where percent homology canbe determined by, for example, BLAST or FASTA software. In certainnon-limiting embodiments, the complementary portion may constitute atleast 10 nucleotides or at least 15 nucleotides or at least 20nucleotides or at least 25 nucleotides or at least 30 nucleotides andthe antisense nucleic acid, shRNA or siRNA molecules may be up to 15 orup to 20 or up to 25 or up to 30 or up to 35 or up to 40 or up to 45 orup to 50 or up to 75 or up to 100 nucleotides in length. Non-limitingexamples of a shRNA that inhibit Cx43 are set forth in the Examplebelow. In non-limiting embodiments, a Cx43 inhibitor, which is a nucleicacid, may be provided in a Cx43-expressing cancer cell via a vector, forexample a lentivirus, which may be selectively targeted to said cancercell and/or wherein expression of the Cx43 inhibitor nucleic acid may bedirected by a promoter which is selectively active in tumor cells.

5.1.2 Protocadherin 7 Inhibitors

The present invention provides Protocadherin 7 (PCDH7) inhibitors foruse in the disclosed methods. Non-limiting examples of PCDH7 inhibitorsinclude compounds, molecules, chemicals, polypeptides, proteins thatinhibit and/or reduce the expression and/or activity of PCDH7. A PCDH7nucleic acid or protein may be a human PCDH7 nucleic acid having thesequence as set forth in NCBI database accession no. NM_001173523,NM_032457, NM_032456 or NM_002589, or a nucleic acid encoding a humanPCDH7 protein molecule that has the amino acid set forth in NCBIdatabase accession no. NP_001166994, NP_115832, NP_115833 or NP_002580.

In certain non-limiting embodiments, PCDH7 inhibitors can includeribozymes, antisense oligonucleotides, shRNA molecules and siRNAmolecules that specifically inhibit and/or reduce the expression oractivity of PCDH7. One non-limiting example of a PCDH7 inhibitorcomprises an antisense, shRNA or siRNA nucleic acid sequence homologousto at least a portion of a PCDH7 nucleic acid sequence, wherein thehomology of the portion relative to the PCDH7 sequence is at least about75 or at least about 80 or at least about 85 or at least about 90 or atleast about 95 or at least about 98 percent, where percent homology canbe determined by, for example, BLAST or FASTA software. In certainnon-limiting embodiments, the complementary portion may constitute atleast 10 nucleotides or at least 15 nucleotides or at least 20nucleotides or at least 25 nucleotides or at least 30 nucleotides andthe antisense nucleic acid, shRNA or siRNA molecules may be up to 15 orup to 20 or up to 25 or up to 30 or up to 35 or up to 40 or up to 45 orup to 50 or up to 75 or up to 100 nucleotides in length. In certainembodiments, antisense, shRNA or siRNA molecules of the presentinvention may comprise DNA or atypical or non-naturally occurringresidues as disclosed above, for example, but not limited to,phosphorothioate residues. Non-limiting examples of a shRNA thatinhibits PCDH7 are set forth in the Example below. In non-limitingembodiments, a PCDH7 inhibitor, which is a nucleic acid, may be providedin a PCDH7-expressing cancer cell via a vector, for example alentivirus, which may be selectively targeted to said cancer cell and/orwherein expression of the PCDH7 inhibitor nucleic acid may be directedby a promoter which is selectively active in tumor cells.

In non-limiting embodiments, a PCDH7 inhibitor can be an antibody orantibody fragment or single chain antibody that specifically binds toPCDH7. Non-limiting examples of such antibodies include ab55506 (AbcamInc.) and HPA011866 (Sigma-Aldrich). In certain non-limitingembodiments, an anti-PCDH7 antibody or antibody fragment may be used toprepare a human, humanized or otherwise chimeric antibody that isspecific for PCDH7 for use according to the invention.

5.1.3 Assay for Gap Junction Activity/Inhibition

Certain non-limiting embodiments of the invention provide for an assayfor evaluating gap junction activity, for example assessing inhibition,by measuring levels of cyclic guanosine monophosphate-adenosinemonophosphate, e.g., [G(2′,5′)pA(3′,5′)p](“cGAMP”), where a decrease incGAMP correlates with gap junction inhibition. This aspect of theinvention is based, at least in part, on the discovery that cGAMPincreases when gap junctions form between astrocytes and cancer cellsthat have metastasized to the brain, and that said elevated cGAMPdecreases with Connexin 43 inhibition (see, for example, FIGS. 4B and14B).

Particular non-limiting embodiments provide for a method for inhibitinggrowth and/or survival of metastatic cancer cells in the brain of asubject, comprising treating the subject with a therapeuticallyeffective amount of a gap junction inhibitor that produces a decrease incGAMP relative to the level of cGAMP in the absence of that amount ofgap junction inhibitor.

Particular non-limiting embodiments provide for a method of determiningwhether a brain tumor or metastatic brain tumor in a subject willreceive therapeutic benefit from treatment with a gap junctioninhibitor, comprising determining whether, in a sample from said tumor,exposure to a gap junction inhibitor leads to a decrease in cGAMP, wherea decrease in cGAMP is indicative of therapeutic benefit.

Further non-limiting embodiments of the invention provide for a methodof inhibiting growth and/or survival of metastatic cancer cells in thebrain of a subject, comprising (i) determining whether the subject willreceive therapeutic benefit from treatment with a gap junctioninhibitor, comprising determining whether cancer cells of the subject(which may be obtained from a brain metastasis, the primary tumor, or ametastatic tumor outside the brain), when exposed to a gap junctioninhibitor, exhibit a decrease in cGAMP relative to the cGAMP level inthe absence of the inhibitor, where a decrease in cGAMP is indicative oftherapeutic benefit; and (ii) where a decrease in cGAMP is observed,treating the subject with the gap junction inhibitor or, where adecrease in cGAMP is not observed, either assaying another gap junctioninhibitor for its ability to decrease cGAMP in the tumor cells ortreating the subject with another modality, such as chemotherapy,immunotherapy, radiation therapy, etc. Said determination may beperformed, for example, using an in vitro assay as described in theworking example below, or a comparable cGAMP measuring system known inthe art.

Further non-limiting embodiments of the invention provide for a methodof inhibiting growth of a brain tumor in a subject, comprising (i)determining whether the subject will receive therapeutic benefit fromtreatment with a gap junction inhibitor, comprising determining whethera tumor cell(s) of the subject, when exposed to a gap junctioninhibitor, exhibits a decrease in cGAMP relative to the cGAMP level inthe absence of the inhibitor, where a decrease in cGAMP is indicative oftherapeutic benefit; and (ii) where a decrease in cGAMP is observed,treating the subject with the gap junction inhibitor or, where adecrease in cGAMP is not observed, either assaying another gap junctioninhibitor for its ability to decrease cGAMP in the tumor cell(s) ortreating the subject with another modality, such as chemotherapy,immunotherapy, radiation therapy, etc. Said determination may beperformed, for example, using an in vitro assay as described in theworking example below, or a comparable cGAMP measuring system known inthe art.

cGAMP may be measured by any method known in the art. In certainnon-limiting embodiments of the invention, a cGAMP level is determinedby Liquid Chromatography Mass Spectrometry/Mass Spectrometry(“LC-MS/MS”). the LC-MS/MS may be normalized to an internal standard(for example, to account for any losses in the purification steps). Asone specific non-limiting example, an assay is described in the workingexample below, section “cGAMP quantitation by LC-MS/MS,” incorporated byreference in this detailed description. See also FIG. 16.

In certain non-limiting embodiments, the present invention provides fora kit to be used in said assay, comprising at least one cGAMP standard,and information regarding decrease of cGAMP with gap junction inhibitionin brain tumors.

In certain non-limiting embodiments, the present invention provides fora kit for detecting the amount of cGAMP present within a sample. Incertain embodiments, a kit can comprise isotopically labeled cGAMP. Forexample, and not by way of limitation, the isotopically labeled cGAMPcan be used as an internal control in analytical chemistry techniques,e.g., mass spectrometry (MS) and Liquid chromatography (LC)-MS/MS. Incertain embodiments, the isotopically labeled cGAMP can be enriched witha low abundance stable isotope such as, but not limited to, 2H(deuterium), 13C (carbon-13), 15N (nitrogen-15) or 180 (oxygen-18).

In certain non-limiting embodiments, a kit of the present invention canfurther include instructions for using the kit to detect the amount ofcGAMP in a sample. For example, and not by way of limitation, theinstructions can describe the amount of isotopically labeled cGAMP toadd to a sample prior to analysis. In certain embodiments, theinstructions can further describe how to calculate the amount of cGAMPin the sample from the amount of isotopically labeled cGAMP added to thesample. In certain non-limiting embodiments, the instructions candescribe that reduction in the amount or level of cGAMP in a sample froma subject in response to a gap junction inhibitor, as compared to areference control level, is indicative of therapeutic benefit from useof the gap junction inhibitor.

5.2 Cancer Targets

In certain embodiments, the present invention provides methods fortreating brain metastasis. “Metastasis,” as used herein, refers to thepresence of one or more cancer cells at a location that is notphysically contiguous with the original location of the cancer (e.g.,primary cancer). For example, and not by way of limitation, the cancercan include lung cancer, breast cancer, melanoma, colon cancer, kidneycancer, renal cell carcinoma, mesothelioma, ovarian cancer, pancreaticcancer, sarcoma, leukemia, lymphoma, urothelial cancer, head and neckcancer, osteosarcoma and bladder cancer. In certain embodiments, thecancer can include glioblastoma and astrocytoma.

A “detectable” metastasis is a cluster of cells that may be identifiableby magnetic resonance imaging, computerized tomography or positronemission tomography. In certain non-limiting embodiments, a cluster ofmetastatic cells may include at least about 1×10⁷ cells. In certainembodiments, a detectable metastasis can include a cluster of cellshaving a size greater than about 5 mm or about 10 mm.

5.3 Pharmaceutical Formulations

In certain non-limiting embodiments, the present invention provides forpharmaceutical formulations of the gap junction inhibitors disclosedabove in section 5.1 for therapeutic use. In certain embodiments, thepharmaceutical formulation comprises a gap junction inhibitor and apharmaceutically acceptable carrier.

“Pharmaceutically acceptable,” as used herein, includes any carrierwhich does not interfere with the effectiveness of the biologicalactivity of the active ingredients, e.g., inhibitors, and that is nottoxic to the patient to whom it is administered. Non-limiting examplesof suitable pharmaceutical carriers include phosphate-buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents and sterile solutions. Additional non-limitingexamples of pharmaceutically acceptable carriers can include gels,bioadsorbable matrix materials, implantation elements containing theinhibitor and/or any other suitable vehicle, delivery or dispensingmeans or material. Such carriers can be formulated by conventionalmethods and can be administered to the subject.

In certain non-limiting embodiments, the pharmaceutical formulations ofthe present invention can be formulated using pharmaceuticallyacceptable carriers well known in the art that are suitable for oraladministration. Such carriers enable the pharmaceutical compositions tobe formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral or nasal ingestion by apatient to be treated. In certain embodiments, the pharmaceuticalformulation can be a solid dosage form. In certain embodiments, thetablet can be an immediate release tablet. Alternatively oradditionally, the tablet can be an extended or controlled releasetablet. In certain embodiments, the solid dosage can include both animmediate release portion and an extended or controlled release portion.In certain embodiments, the pharmaceutical formulations of the presentinvention can be formulated using pharmaceutically acceptable carrierswell known in the art that are suitable for parenteral administration.

In certain embodiments, the pharmaceutical formulations suitable for usein the present invention can include formulations where the activeingredients, e.g., gap junction inhibitors, are contained in atherapeutically effective amount. A “therapeutically effective amount”refers to an amount that is able to achieve one or more of ananti-cancer effect, prolongation of survival and/or prolongation ofperiod until relapse. The therapeutically effective amount of an activeingredient can vary depending on the active ingredient, e.g., gapjunction inhibitor, formulation used, the cancer and its severity, andthe age, weight, etc., of the subject to be treated. In certainembodiments, a patient can receive a therapeutically effective amount ofa gap junction inhibitor in single or multiple administrations of one ormore formulations, which can depend on the dosage and frequency asrequired and tolerated by the patient.

An “anti-cancer effect” or “therapeutic benefit” as used herein, refersto one or more of a reduction in aggregate cancer cell mass, a reductionin cancer cell growth rate, a reduction in cancer cell proliferation, areduction in tumor mass, a reduction in tumor volume, a reduction intumor cell proliferation, a reduction in tumor growth rate and/or areduction in tumor metastasis. In certain embodiments, an anti-cancereffect can refer to a complete response, a partial response, a stabledisease (without progression or relapse) and/or a response with a laterrelapse or progression-free survival in a patient diagnosed with cancer.In certain embodiments, an anti-cancer effect can refer to theprevention and/or reduction of metastasis of a primary cancer within asubject, e.g., the prevention and/or reduction of metastasis of a cancerto the brain in a subject.

In certain non-limiting embodiments, the gap junction inhibitorsdescribed above can be used alone or in combination with one or moreanti-cancer agents. An “anti-cancer agent,” as used herein, can be anymolecule, compound, chemical or composition that has an anti-cancereffect. Anti-cancer agents include, but are not limited to,chemotherapeutic agents, radiotherapeutic agents, cytokines,anti-angiogenic agents, apoptosis-inducing agents, anti-cancerantibodies, anti-cyclin-dependent kinase agents and/or agents whichpromote the activity of the immune system including, but not limited to,cytokines such as but not limited to interleukin 2, interferon,anti-CTLA4 antibody and/or anti-PD-1 antibody. Non-limiting examples ofanti-cancer agents include paclitaxel, temozolomide, vinorelbine,procarbazine, lomustine, vincristine, sFasL and carboplatin. Forexample, but not by way of limitation, a gap junction inhibitor, e.g.,meclofenamate and/or tonabersat, can be used in combination withcarboplatin. “In combination with,” as used herein, means that the gapjunction inhibitor and the one or more anti-cancer agents areadministered to a subject as part of a treatment regimen or plan. Incertain embodiments, being used in combination does not require that theinhibitor and one or more anti-cancer agents are physically combinedprior to administration or that they be administered over the same timeframe.

In certain embodiments, where an inhibitor is used in combination withan anti-cancer agent, the amount of each may in some instances be lessthan a therapeutically effective amount for that agent taken singly, butwhen both are used therapeutically effectiveness is achieved.

5.4 Methods of Treatment

The present invention relates to methods for treating brain metastasisby inhibiting gap junction functionality. As described in detail in theExample section below, the studies presented in the instant applicationindicate that inhibition of gap junction signaling and/or formationbetween the cancer cell and astrocyte can be used to treat brainmetastasis. It is based, at least in part, on the discovery that cancercells expressing Protocadherin 7 and Connexin 43 form gap junctions withastrocytes, which promote the growth of brain metastases, and thatinhibition of Protocadherin 7 and/or Connexin 43 expression in cancercells reduce progression of brain metastases. It is further based on thediscovery that treatment with gap junction inhibitors tonabersat andmeclofenamate inhibited progression of brain metastatic lesions andenhanced the anti-cancer activity of the conventional chemotherapeuticagent, carboplatin.

Accordingly, the present invention provides methods of treating brainmetastasis by inhibiting gap junction signaling and/or formation by theadministration of a gap junction inhibitor, disclosed above.Non-limiting examples of gap junction inhibitors, and pharmaceuticalformulations thereof, are disclosed in sections 5.1 and 5.3, above.Cancers that can be treated with the methods of the present inventionare disclosed above in section 5.2. As such, the present inventionrelates to methods for inhibiting gap junction functionality to producean anti-cancer effect in a subject.

A “subject” or “patient,” as used interchangeably herein, refers to ahuman or a non-human subject. Non-limiting examples of non-humansubjects include non-human primates, dogs, cats, mice, rats, guineapigs, rabbits, pigs, fowl, horses, cows, goats and sheep.

In certain non-limiting embodiments, the present invention provides fora method of treating a subject having a cancer comprising administering,to the subject, an amount of a gap junction inhibitor that inhibitsmetastatic progression of the cancer in the brain. In certainembodiments, the gap junction inhibitor can be meclofenamate,tonabersat, a Cx43 inhibitor and/or a PCDH7 inhibitor. In certainembodiments, the cancer can be breast cancer. In certain embodiments,the cancer can be lung cancer. In certain non-limiting embodiments, oneor more cells of the cancer of the subject express Connexin 43 and/orProtocadherin 7. In certain embodiments, the subject was known to haveone or more brain metastases prior to treatment. In certain non-limitingembodiments of the present invention, the subject was not known to havea brain metastasis prior to treatment.

In certain embodiments, the method of treating a subject having a cancercomprises administering, to the subject, an amount of tonabersat toinhibit metastatic progression of the cancer in the brain.

In certain embodiments, the method of treating a subject having a cancercomprises administering, to the subject, an amount of meclofenamate toinhibit metastatic progression of the cancer in the brain.

In certain embodiments, the method of treating a subject having a cancercomprises administering, to the subject, an amount of a Cx43 inhibitorto inhibit metastatic progression of the cancer in the brain.

In certain embodiments, the method of treating a subject having a cancercomprises administering, to the subject, an amount of a PCDH7 inhibitorto inhibit metastatic progression of the cancer in the brain.

In certain non-limiting embodiments, the present invention furtherprovides for a method for inhibiting growth and/or survival ofmetastatic cancer cells in the brain of a subject, comprisingadministering, to the subject, a therapeutically effective amount of agap junction inhibitor, disclosed above. In certain embodiments, the gapjunction inhibitor can be meclofenamate, tonabersat, a Cx43 inhibitorand/or a PCDH7 inhibitor. In certain embodiments, the cancer is lungcancer and/or breast cancer. In certain non-limiting embodiments, one ormore cells of the cancer of the subject express Connexin 43 and/orProtocadherin 7. In certain embodiments, the subject was known to haveone or more brain metastases prior to treatment. In certain non-limitingembodiments of the present invention, the subject was not known to havea brain metastasis prior to treatment.

In certain embodiments, the method for inhibiting growth and/or survivalof metastatic cancer cells in the brain of a subject comprisesadministering, to the subject, a therapeutically effective amount oftonabersat.

In certain embodiments, the method for inhibiting growth and/or survivalof metastatic cancer cells in the brain of a subject comprisesadministering, to the subject, a therapeutically effective amount ofmeclofenamate.

In certain embodiments, the method for inhibiting growth and/or survivalof metastatic cancer cells in the brain of a subject comprisesadministering, to the subject, a therapeutically effective amount of aCx43 inhibitor.

In certain embodiments, the method for inhibiting growth and/or survivalof metastatic cancer cells in the brain of a subject comprisesadministering, to the subject, a therapeutically effective amount of aPCDH7 inhibitor.

In certain non-limiting embodiments, the present invention provides fora method of treating brain metastasis in a subject comprisingadministering, to the subject, a therapeutically effective amount of agap junction inhibitor, disclosed above. In certain non-limitingembodiments, the gap junction inhibitor can be meclofenamate,tonabersat, a Cx43 inhibitor and/or a PCDH7 inhibitor. In certainembodiments, the cancer is lung cancer and/or breast cancer. In certainnon-limiting embodiments, one or more cells of the cancer of the subjectexpress Connexin 43 and/or Protocadherin 7. In certain embodiments, thebrain metastasis is a detectable metastasis.

In certain embodiments, the method of treating brain metastasis in asubject comprises administering, to the subject, a therapeuticallyeffective amount of tonabersat.

In certain embodiments, the method of treating brain metastasis in asubject comprises administering, to the subject, a therapeuticallyeffective amount of meclofenamate.

In certain embodiments, the method of treating brain metastasis in asubject comprises administering, to the subject, a therapeuticallyeffective amount of a Cx43 inhibitor.

In certain embodiments, the method of treating brain metastasis in asubject comprises administering, to the subject, a therapeuticallyeffective amount of a PCDH7 inhibitor.

In certain non-limiting embodiments, the present invention provides fora method of preventing metastasis of a cancer to the brain in a subjectcomprising administering, to the subject, a therapeutically effectiveamount of a gap junction inhibitor, disclosed above. In certainembodiments, the gap junction inhibitor can be meclofenamate,tonabersat, a Cx43 inhibitor and/or a PCDH7 inhibitor. In certainembodiments, the cancer is lung cancer and/or breast cancer. In certainnon-limiting embodiments, one or more cells of the cancer of the subjectexpress Connexin 43 and/or Protocadherin 7. In certain non-limitingembodiments of the present invention, the subject was not known to havea brain metastasis prior to treatment.

In certain embodiments, the method of preventing metastasis of a cancerto the brain in a subject comprises administering, to the subject, atherapeutically effective amount of tonabersat.

In certain embodiments, the method of preventing metastasis of a cancerto the brain in a subject comprises administering, to the subject, atherapeutically effective amount of meclofenamate.

In certain embodiments, the method of preventing metastasis of a cancerto the brain in a subject comprises administering, to the subject, atherapeutically effective amount of a Cx43 inhibitor.

In certain embodiments, the method of preventing metastasis of a cancerto the brain in a subject comprises administering, to the subject, atherapeutically effective amount of a PCDH7 inhibitor.

In certain non-limiting embodiments, the present invention provides fora method of reducing the risk of detectable metastasis of a cancer tothe brain in a subject having cancer comprising administering, to thesubject, a therapeutically effective amount of a gap junction inhibitor,disclosed above. In certain embodiments, the gap junction inhibitor canbe meclofenamate, tonabersat, a Cx43 inhibitor and/or a PCDH7 inhibitor.

In certain embodiments, the cancer is lung cancer and/or breast cancer.In certain non-limiting embodiments, one or more cells of the cancer ofthe subject express Connexin 43 and/or Protocadherin 7. In certainembodiments, the subject was known to have one or more brain metastasesprior to treatment. In certain non-limiting embodiments of the presentinvention, the subject was not known to have a brain metastasis prior totreatment.

In certain embodiments, the method of reducing the risk of detectablemetastasis of a cancer to the brain in a subject having cancer comprisesadministering, to the subject, a therapeutically effective amount oftonabersat.

In certain embodiments, the method of reducing the risk of detectablemetastasis of a cancer to the brain in a subject having cancer comprisesadministering, to the subject, a therapeutically effective amount ofmeclofenamate.

In certain embodiments, the method of reducing the risk of detectablemetastasis of a cancer to the brain in a subject having cancer comprisesadministering, to the subject, a therapeutically effective amount of aCx43 inhibitor.

In certain embodiments, the method of reducing the risk of detectablemetastasis of a cancer to the brain in a subject having cancer comprisesadministering, to the subject, a therapeutically effective amount of aPCDH7 inhibitor.

In certain embodiments, the present invention provides a method forlengthening the period of survival of a subject having a cancercomprising administering, to the subject, a therapeutically effectiveamount of a gap junction inhibitor, disclosed above. In certainembodiments, the gap junction inhibitor can be meclofenamate,tonabersat, a Cx43 inhibitor and/or a PCDH7 inhibitor. In certainembodiments, the cancer is lung cancer and/or breast cancer. In certainnon-limiting embodiments, one or more cells of the cancer of the subjectexpress Connexin 43 and/or Protocadherin 7. In certain embodiments, thesubject was known to have one or more brain metastases prior totreatment. In certain non-limiting embodiments of the present invention,the subject was not known to have a brain metastasis prior to treatment.

In certain embodiments, the method for lengthening the period ofsurvival of a subject having a cancer comprises administering, to thesubject, a therapeutically effective amount of tonabersat.

In certain embodiments, the method for lengthening the period ofsurvival of a subject having a cancer comprises administering, to thesubject, a therapeutically effective amount of meclofenamate.

In certain embodiments, the method for lengthening the period ofsurvival of a subject having a cancer comprises administering, to thesubject, a therapeutically effective amount of a Cx43 inhibitor.

In certain embodiments, the method for lengthening the period ofsurvival of a subject having a cancer comprises administering, to thesubject, a therapeutically effective amount of a PCDH7 inhibitor.

In certain embodiments, the methods of the present invention canlengthen the survival period of a subject having cancer by about 1month, about 2 months, about 3 months, about 4 months, about 6 months,about 8 months, about 10 months, about 12 months, about 14 months, about18 months, about 20 months, about 2 years, about 3 years, about 4 years,about 5 years, about 6 years or more.

In certain embodiments, a method for treating cancer cell metastasis ina subject in need of such treatment comprises administering, to thesubject, a therapeutically effective amount of a gap junction inhibitor,disclosed above, to inhibit cancer cell-astrocyte gap junctionfunctionality.

In certain embodiments, the present invention provides a method ofproducing an anti-cancer effect in a subject having a cancer comprisingadministering, to the subject, a therapeutically effective amount of agap junction inhibitor, disclosed above.

In certain embodiments, the present invention provides a method ofproducing an anti-cancer effect in a subject having a cancer comprisingadministering, to the subject, a therapeutically effective amount of agap junction inhibitor, disclosed above, to inhibit cancercell-astrocyte gap junction functionality.

In certain embodiments, the present invention provides a method ofproducing an anti-cancer effect in a subject having a cancer comprisingadministering, to the subject, a therapeutically effective amount of agap junction inhibitor to inhibit gap junction functionality.

In certain embodiments, the present invention provides methods fortreating a subject that has cancer, for inhibiting the growth and/orsurvival of cancer cells, for preventing and/or delaying thereoccurrence of a cancer, for inhibiting the infiltration of cancercells and for lengthening the period of survival of a subject havingcancer, comprising, administering, to the subject, a therapeuticallyeffective amount of a gap junction inhibitor, disclosed above. Incertain embodiments, the cancer is glioblastoma and/or astrocytoma.

In certain embodiments, the methods of the present invention can furthercomprise administering to the subject an anti-cancer agent, as describedabove. For example, and not by way of limitation, a method of thepresent invention comprises administering, to the subject, atherapeutically effective amount of a gap junction inhibitor and atherapeutically effective amount of an anti-cancer agent that canpenetrate the blood brain barrier to achieve therapeutic levels, suchas, but not limited to ACNU, BCNU, CCNU, hydroxyurea, topotecan,temozolomide, dacarbazine, methotrexate, Ara-C, capecitabine, cisplatin,vinorelbine, carboplatin, or combinations thereof.

In certain embodiments, a method of the present invention comprisesadministering, to the subject, a therapeutically effective amount ofmeclofenamate and a therapeutically effective amount of carboplatin.

In certain embodiments, a method of the present invention comprisesadministering, to the subject, a therapeutically effective amount oftonabersat and a therapeutically effective amount of carboplatin.

In certain embodiments, a method of the present invention comprisesadministering, to the subject, a therapeutically effective amount of aCx43 inhibitor and a therapeutically effective amount of carboplatin.

In certain embodiments, a method of the present invention comprisesadministering, to the subject, a therapeutically effective amount of aPCDH7 inhibitor and a therapeutically effective amount of carboplatin.

In a specific non-limiting embodiment, a gap junction inhibitor can beadministered at an amount of about 1 mg/kg to about 30 mg/kg. Forexample, and not by way of limitation, a gap junction inhibitor can beadministered at an amount of about 1 mg/kg to about 25 mg/kg, about 1mg/kg to about 20 mg/kg, about 1 mg/kg to about 15 mg/kg, about 1 mg/kgto about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 5 mg/kg toabout 30 mg/kg, about 10 mg/kg to about 30 mg/kg, about 15 mg/kg toabout 30 mg/kg, about 20 mg/kg to about 30 mg/kg or about 25 mg/kg toabout 30 mg/kg. In certain non-limiting embodiments, the gap junctioninhibitor can be administered at an amount of about 0.08 mg/kg to about3.6 mg/kg (see Reagan-Shaw et al., The FASEB J., Vol. 22: 659-661(2008)). In certain non-limiting embodiments, the gap junction inhibitorcan be administered at an amount of about 0.15 mg/kg to about 18 mg/kg.

In certain non-limiting embodiments, the gap junction inhibitor can beadministered at an amount of about 1 mg to about 200 mg. For example,and not by way of limitation, a gap junction inhibitor can beadministered at an amount of about 1 mg to about 200 mg, about 10 mg toabout 200 mg, about 20 mg to about 200 mg, about 30 mg to about 200 mg,about 40 mg to about 200 mg, about 50 mg to about 200 mg, about 60 mg toabout 200 mg, about 70 mg to about 200 mg, about 80 mg to about 200 mg,about 90 mg to about 200 mg, about 100 mg to about 200 mg, about 110 mgto about 200 mg, about 120 mg to about 200 mg, about 130 mg to about 200mg, about 140 mg to about 200 mg, about 150 mg to about 200 mg, about160 mg to about 200 mg, about 170 mg to about 200 mg, about 180 mg toabout 200 mg, about 190 mg to about 200 mg, about 1 mg to about 190 mg,about 1 mg to about 180 mg, about 1 mg to about 170 mg, about 1 mg toabout 160 mg, about 1 mg to about 150 mg, about 1 mg to about 140 mg,about 1 mg to about 130 mg, about 1 mg to about 120 mg, about 1 mg toabout 110 mg, about 1 mg to about 100 mg, about 1 mg to about 90 mg,about 1 mg to about 80 mg, about 1 mg to about 70 mg, about 1 mg toabout 60 mg, about 1 mg to about 50 mg, about 1 mg to about 40 mg, about1 mg to about 30 mg about 1 mg to about 20 mg, about 1 mg to about 10 mgor about 1 mg to about 5 mg.

In certain embodiments, the gap junction inhibitor tonabersat can beadministered at an amount of about 10 mg/kg. In certain embodiments, thegap junction inhibitor tonabersat can be administered at an amount ofabout 0.8 mg/kg to about 1.2 mg/kg. In certain embodiments, the gapjunction inhibitor tonabersat can be administered at an amount of about0.01 mg/kg to about 9 mg/kg. In certain embodiments, the gap junctioninhibitor meclofenamate can be administered at an amount of about 20mg/kg. In certain embodiments, the gap junction inhibitor meclofenamatecan be administered at an amount of about 1.6 mg/kg to about 2.4 mg/kg.In certain embodiments, the gap junction inhibitor meclofenamate can beadministered at an amount of about 0.1 mg/kg to about 19 mg/kg. Incertain embodiments, the gap junction inhibitor meclofenamate can beadministered at an amount of between about 100 mg to about 400 mg daily.In certain embodiments, the gap junction inhibitor meclofenamate can beadministered at an amount of about 100 mg twice daily. In certainembodiments, a subject is treated concurrently with a proton-pumpinhibitor and meclofenamate. In certain embodiments, the gap junctioninhibitor meclofenamate can be administered at an amount of about 100 mgtwice daily, the subject may be treated concurrently with a proton-pumpinhibitor and meclofenamate, and the treatment period may be at leastabout 2 months, at least about 4 months, or at least about 6 months.

In a specific non-limiting embodiment, an anti-cancer agent can beadministered at an amount of about 1 nM to about 1 μM and/or about 10mg/kg to about 100 mg/kg. In a specific non-limiting embodiment, ananti-cancer agent can be administered at an amount of about 0.8 mg/kg toabout 8 mg/kg. In a specific non-limiting embodiment, an anti-canceragent can be administered at an amount of about 1.2 mg/kg to about 60mg/kg. For example, and not by way of limitation, the anti-cancer agentcarboplatin can be administered at an amount of about 500 nM and/orabout 50 mg/kg. In certain embodiments, the anti-cancer agentcarboplatin can be administered at an amount of about 4 to about 6mg/kg. In certain embodiments, the anti-cancer agent Paclitaxel can beadministered at an amount of about 25 nM.

In certain embodiments, the gap junction inhibitors of the presentinvention can be administered once, twice, three, four, five or sixtimes per week, or daily. In certain embodiments, the anti-cancer agentsof the present invention can be administered once, twice, three, four,five, or six times per week, or daily. In certain embodiments, theinhibitors and/or anti-cancer agents of the presently disclosed subjectmatter can be administered one or more times per day. For example, andnot by way of limitation, the gap junction inhibitors and/or anti-canceragents of the present invention can be administered once, twice, three,four, five or more times a day.

An inhibitor and/or an anti-cancer agent, disclosed herein, can beadministered to the subject using standard methods of administration. Incertain embodiments, the inhibitor can be administered to the subjectorally or parenterally. For example, and not by way of limitation, theroute of administration can be intravenous, intraarterial, intrathecal,intraperitoneal, intramuscular, subcutaneous, topical, intradermal,locally or combinations thereof. In certain embodiments, the inhibitorcan be administered to the patient from a source implanted in thepatient. In certain embodiments, administration of the inhibitor canoccur by continuous infusion over a selected period of time.

The following example is merely illustrative of the presently disclosedinvention and should not be considered as a limitation in any way.

6. EXAMPLE 1: PROTOCADHERIN 7 AND CONNEXIN 43 MEDIATECARCINOMA-ASTROCYTE GAP JUNCTIONS AND BRAIN METASTASIS 6.1 Materials andMethods

Cell culture.

Human MDA-MB-231 (MDA231), murine MMTV-neu, their metastaticderivatives, and murine 373N1, 393N1, 482N1, 2691N1 cell lines werecultured in DMEM with 10% fetal bovine serum (FBS) and 2 mM L-Glutamine.Human H2030 cells and metastatic derivatives were cultured in RPMI 1640medium supplemented with 10% FBS and 2 mM L-Glutamine. For lentivirusproduction, 293T cells were cultured in DME media supplemented with 10%fetal bovine serum and 2 mM L-glutamine. Human primary astrocytes, brainmicrovascular endothelial cells (HBMEC), adult dermal fibroblasts, andmicroglia were cultured in media specified by the supplier (ScienCell),and used between passages 2-6. All cells tested negative for micoplasma.

Animal Studies.

All experiments using animals were done in accordance to protocolsapproved by the MSKCC Institutional Animal Care and Use Committee.Athymic NCR nu/nu mice (NCI-Frederick), Cr:NIH bg-nu-xid mice(NCI-Frederick) and B6129SF1/J mice (Jackson Laboratory) were use at 5-6weeks of age. For long-term brain metastasis assays we followedpreviously described procedures (Bos, Nguyen et al. 2010). In brief, 10⁴MDA231-BrM2 cells, 5×10⁴ H2030-BrM3 cells, or 10⁵ 393N1 cells suspendedin 100 μl of PBS were injected into the left cardiac ventricle. At theexperimental endpoint, anesthetized mice (ketamine 100 mg/kg, xylazine10 mg/kg) were injected retro-orbitally with D-luciferin (150 mg/kg),and brain colonization was quantified by ex vivo Bio-luminescent imaging(BLI). For short-term (7-day and 14-day) brain metastasis experiments,we injected 5×10⁵ cells. TRITC dextran (70 KD) (Life Technologies) wasintravenously injected to stain vascular structures. For inducibleknockdown experiments, mice were given doxycycline hyclate(Sigma-Aldrich) in the drinking water (2 mg/mL) and the diet (Harlan) 14days after injection of cancer cells. For lung colonization assays,2×10⁵ MDA231-BrM2 cells in 100 piL PBS were injected into the lateraltail vein. For orthotopic tumour implantation, 5×10³ cells in 50 piL of1:1 mix of PBS/growth factor reduced matrigel (BD Biosciences) wereinjected into the 4th right mammary fat pad of female mice. For drugtreatment experiments, mice were intraperitoneally injected withcarboplatin (Hospira) (50 mg/kg/5 days), Tonabersat (MedChem Express)(10 mg/kg/day), or meclofenamic acid sodium salt (Sigma-Aldrich) (20mg/kg/day). Vehicle (10% DMSO in Polyethylene glycol 400) was used incontrol mice. BLI was performed using an IVIS Spectrum Xenogeninstrument (Caliper Life Sciences) and analysed using Living Imagesoftware, v. 2.50. For brain metastasis assays, 8-10 mice were used ineach group. For drug treatment experiments, mice were inoculated withcancer cells and randomly assigned to treatment groups. Gap junctionmodulators and chemotherapeutic agents were blindly administered in theMSKCC Antitumour Assessment Core.

Knockdown and Overexpression Constructs.

For stable knockdown of Cx43 and PCDH7, we used shRNAs in lentiviralvectors. For inducible knockdown, shRNAs in TRIPZ lentivial vector wereused. 1 μg/mL doxycycline hyclate (Sigma-Aldrich) was added to inducethe expression of shRNA. Targeted sequences of shRNAs are listed Table1, below. pBabe-Puro-IKBalpha-mut (Addgene) was used for stableexpression of SR-IkB. For expression of wild type Cx43 (Origene), orCx43(T154A) mutant (ACC to GCC), we used pLVX vector.

mRNA and Protein Detection.

Total RNA was extracted using the PrepEase RNA spin kit (USB). Toprepare cDNA, 1 μg of total RNA was treated using the Transcriptor FirstStrand cDNA synthesis kit (Roche). Cx43, Cx30 and Cx 26 expression wasquantified by Taqman gene expression assay primers: (Cx 43:Hs00748445_s1, Mm00439105_ml; Cx30: Hs00922742_s1, Mm00433661_s1; Cx26:Hs00269615_s1, Mm00433643_s1; Applied Biosystems). Relative geneexpression was normalized relative to β2-microglobulin (Hs99999907_m1,Mm00437762_m1). The PCDH7 primer pair was designed to detect all PCDH7isoforms: 5′-agttcaacgtggtcatcgtg-3′(sense),5′-acaatcagggagttgttgctc-3′(antisense). Reactions were performed usingSYBR Green I Master Mix (Applied Biosystems). Quantitative expressiondata were analyzed using an ABI Prism 7900HT Sequence Detection System(Applied Biosystems). For western immunoblotting, cell pellets werelysed with RIPA buffer and protein concentrations determined by BCAProtein Assay Kit (Pierce). Protein lysates of primary human astrocytes,neurons, microglia and HBMEC were purchased from ScienCell. Proteinswere separated by SDS-PAGE and transferred to nitrocellulose membranes(BioRad). Antibodies used for western blotting are listed in Table 2,below.

Dye Transfer and EdU Transfer Assays.

Monolayers of cancer cells or astrocytes were labeled with 2.5 gig/mlcalcein Red-Orange AM dye (Life Technologies) at 37° C. for 30 min.Single cell suspensions were mixed at a ratio of 2:1 labeled:unlabeledcells for 6 h. Certain experiments used a mix of three cell populations,MDA231-BrM2 (GFP+), HBMEC (pre-labeled with Cell Proliferating DyeFluor@670, eBioscience), and unlabeled astrocytes. Dye transfer wasvisualized by Zeiss LSM 5 Live confocal microscopy (20-min time-lapse)or quantified by FACSCalibur flow cytometry (BD Biosciences) atdifferent time points. For DNA transfer assays, cancer cells werelabeled overnight with EdU (10 μM, Molecular Probes) and maintained inculture for additional 3 days. Single cell suspensions of labeled cancercells and astrocytes were mixed at 2:1 ratio for 6 h. EdU transfer wasvisualized using Zeiss LSM 5 Live confocal microscopy or quantified byFACSCalibur flow cytometry (BD Biosciences) following the manufacturer'sinstructions (Molecular Probes).

Cancer Cell and Astrocyte Co-Culture Experiments.

Astrocytes and cancer cells were mixed at ratio of 1:1. For apoptosisassays, overnight co-cultures were treated with 500 ng/ml sFasL(Peprotech) in serum free media, 500 nM carboplatin (Sigma-Aldrich) or25 nM Paclitaxel (Sigma-Aldrich) for 24 h. Single cell suspensions werestained with APC-conjugated cleaved caspase 3 antibody (Cell Signaling),apoptotic GFP+ cancer cells were detected by flow cytometery. Fortranslating ribosome affinity purification (TRAP), EGFP-L10a expressingcancer cells were co-cultured with astrocytes for 24 h. Followingpreviously published protocols, (Heiman, Schaefer et al. 2008, Zhang,Jin et al. 2013) mRNA purified from cancer cells were was used forlibrary construction with TruSeq RNA Sample Prep Kit v2 (Illumina)following the manufacturer's instructions. Samples were barcoded and runon a Hiseq 2000 platform in a 50 bp/50 bp paired-end run, using theTruSeq SBS Kit v3 (Illumina). An average of 50 million paired reads weregenerated per sample. For conditioned media analysis, media werecollected after 24 h, and cytokines in the conditioned media were eitheridentified using Human Cytokine Array (R&D systems) or measured by IFNαor TNFα ELISA kits (R&D systems). To detect the activity of IFNα or TNFαin the collected conditioned media, cancer cells were treated with thecollected conditioned media for 2 h and phosphorylation status of STAT1or NF-κB p65 was determined by western blotting. For cGAMP and TBI-IRF3activation experiments, cancer cells and astrocytes were co-cultured for18 h. The phosphorylation status of TBK1, IRF3 was determined by westernimmunoblotting. Nuclear translocation of IRF3 was determined byimmunofluorescence staining with Zeiss LSM 5 Live confocal microscopy.cGAMP levels were determined by LC-MS/MS.

Cytokine Treatment and Pathway Reporter Assays.

Cancer cells were treated with 10 units/ml (39 u/ng) recombinant IFNαA(R&D Systems) or 10 μg/ml recombinant TNFα (R&D Systems) in combinationwith carboplatin or Taxol (Sigma-Aldrich) for 24 h. Apoptosis wasquantified by Caspase-Glo 3/7 assay (Promega). For NFκB reporter assays,the NF-κB responsive sequence from the pHAGE NFKB-TA-LUC-UBC-dTomato-Wconstruct (Addgene)(Wilson, Kwok et al. 2013) was cloned into a pGL4.82Renilla luciferase reporter (Promega). Cancer cells were co-transfectedwith this vector and a LeGO-C2 mCherry vector (Addgene). Renillaluciferase activity was determined using RenillaGlo Luciferase system(Promega). Red fluorescence signal was used to normalize transfectionefficiency.

Immunohistochemical Staining.

Mouse brains were fixed with 4% paraformaldehyde, sectioned by vibratome(Leica) or cryostat (Leica) and stained following established protocols(Valiente, Obenauf et al. 2014). For brain slice assays (Valiente,Obenauf et al. 2014), 250 μm thick slices of adult mouse brain wereprepared with a vibratome (Leica) and placed on top of 0.8 μm poremembranes (Millipore) in brain slice culture medium (DMEM, completeHBSS, 5% FBS, 1 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mLstreptomycin). 3×10⁵ cancer cells were placed on the surface of theslice. After 48 h of incubation, brain slices were fixed with 4%paraformaldehyde, and stained. For immunostaining in chamber slidecultures, cells were fixed with 4% paraformaldehyde and stained.Antibodies used for immunochemical staining are listed in Table 2.Images were acquired with Zeiss Axio Imager.Z1 microscope or Leica SP5upright confocal microscope, and analyzed with ImageJ, Imaris andMetamorph softwares. Antibodies used for immunostaining are listed inTable 2.

Split Luciferase Assay.

Fusion cDNAs were generated by deleting the stop codon in human Cx43(Origene), PCDH7 (Origene), E-cadherin (Addgene) or N-cadherin (Addgene)cDNAs and splicing the N-terminal or C-terminal fragment of fireflyluciferase (Luker, Smith et al. 2004). (Addgene). Constructs were clonedinto pLVX lentiviral expression vector and transduced intonon-GFP-luciferase-labeled parental MDA-MB-231 or H2030 cells. To detectluciferase activity, 7.5 mg/ml D-luciferin potassium salt was added inthe culture media. BLI was performed by IVIS Spectrum Xenogeninstrument, using Living Image software v.2.50.

Cytosolic dsDNA Detection.

For visualization of dsDNA, cells were immunostained with anti-dsDNAantibody. Anti-GFP staining was used to delineate cancer cell bodies,DAPI to distinguish nuclei, and anti-CoxIV antibody (a mitochondrialmarker) to distinguish mitochondria. Phalloidin staining (MolecularProbe) was used to delineate astrocyte cell bodies. For quantificationof dsDNA, nuclear, cytosolic and mitochondrial fractions were preparedusing a mitochondria isolation kit (Thermo Scientific). DNA from allsubcellular fractions was purified by QIAamp DNA mini kit (Qiagen) andquantified by QuantoFluor dsDNA system (Promega).

Bioinformatic and Statistical Analysis.

Bioinformatic analysis was performed in R (ver. 3.1.2) unless otherwisenoted. The data were analyzed using the TopHat2-HTSeq-DESeq2 pipeline(Anders, McCarthy et al. 2013, Kim, Pertea et al. 2013, Love, Huber etal. 2014). Differential gene expression was compared with cooksCutoffand independentFiltering turned off. Scatter plot showing fold changeswas produced using the ggplot2 package. Principal component analysis(PCA) was performed usingprcomp.

Pathway gene response signatures were analyzed and scored by the sum ofz-score method (Zhang, Jin et al. 2013), as previously described(Nguyen, Chiang et al. 2009, Gatza, Lucas et al. 2010). Multiplehypothesis testing was adjusted using the Benjamini & Hochbergfalse-discovery-rate method. Statistical analysis was performed usingGraphPad software (Prism) and Student's t-test (two-tailed). P values<0.05 were considered statistically significant. Values areaverages±standard error of the mean (S.E.M.).

Clinical Sample Analysis.

CX43 and PCDH7 transcript levels were analyzed in the microarray data ofprimary breast cancer (EMC-MSK) and adenocarcinoma datasets (MSKCC set2,GSE3141 and GSE8893). Multiple probes mapping to the same gene werecombined by selecting the probe with maximal variance across samples.Triple-negative breast cancer subtypes were identified either based onclinical annotation of the data set or on ESR1 and ERBB2 transcriptlevels. The hazard ratio of the CX43 and PCDH7 values was computed basedon Cox proportional hazards model, as implemented by the “coxph” commandin R. P values were calculated from a Cox proportional hazard model,with CX43 and PCDH7 expression treated as a continuous variable. ForCx43 immunohistochemistry, normal lung tissue array (75 cases), primarytriple negative breast cancer tissue array (98 cases) and primarynon-small cell lung carcinoma tissue array (138 cases) were purchasedfrom US Biomax. Paraffin embedded tissue microarrays from brainmetastases (117 case of triple-negative breast cancer, 91 cases ofnon-small cell lung carcinoma) were obtained from the MSKCC Departmentof Pathology in compliance with the MSKCC Institutional Review Board.Informed consent was obtained from all subjects. Immunohistochemicalstaining for Cx43 was performed by the MSKCC Pathology Core Facilityusing standardized, automated protocols. For matched primary-brainmetastatic lesions, Cx43 staining images was quantified by positivestaining area (Metamorph software).

cGAMP Quantitation by LC-MS/MS.

Cells (2.4 million MDA231-BRM2 or Human Astrocytes alone, 2.4 millionHuman Astrocytes+2.4 million MDA231-BRM2 co-culture) were seeded in 10cm dishes. After 18 h culture media was aspirated and replaced with 2 mL80:20 methanol:water containing 4 nM c-di-GMP internal standard. Disheswere incubated at −80° C. overnight to promote protein precipitation,scraped and transferred to 2 mL centrifuge tubes. Samples were subjectedto 2 vortex, freeze/thaw cycles in liquid nitrogen, sonicated in an icewater bath at full power for 5 min, and clarified by centrifugation at21,000×g for 20 min at 4° C. Extracts were dried using a bench topevaporator (Genevac) and reconstituted in 100 μL of 0.1% formic acid inwater. Liquid chromatography separation was performed using a ShimadzuHPLC, Accela Open autosampler (Thermo) and Cortecs C18+ column (Waters,150 mm×2.1 mm, 2.7 μm). Samples were maintained at 4° C. and injectionvolume was 15 μL. The aqueous mobile phase (A) was 0.1% formic acid inwater and the organic mobile phase (B) was 0.1% formic acid inacetonitrile. Initial conditions were 0% B with gradient program: 1.0min: 0% B; 7 min: 20% B; 7.1 min: 90% B; 9.0 min: 90% B and 5 minre-equilibration time. Flow rate was 400 μL/min, with a post-columnsolvent of 90:10 acetone:DMSO added to the LC stream using a zero-deadvolume tee at 120 μL/min to boost detection sensitivity. Cyclicnucleotides were detected using a TSQ Vantage mass spectrometer (Thermo)operating in SRM and positive ionization modes. Source parameters were:spray voltage: 4000 V; vaporizer temperature: 200° C., sheath gaspressure: 70 psi; aux gas pressure: 20 psi, capillary temperature: 400°C. Compound-specific S-lens values were: 164 V (cGAMP) and 190 V(c-di-GMP). Individual reactions monitored and collision energies were:cGAMP m/z 675.1→m/z 512.1 (CE: 19 V), m/z 312.0 (CE: 40 V), m/z 136.0(CE: 39 V)* and c-di-GMP m/z 675.1→m/z 540.1 (CE: 19 V), m/z 248.0 V(CE: 27 V), m/z 152.0 (CE: 31 V)*, * indicating the primary transitionused to quantify each cyclic nucleotide. Retention times and transitionswere confirmed relative to cyclic [G(2′,5′)pA(3′,5′)p] and c-di-GMPmetabolite standards (BioLog). Data analysis was performed usingXcalibur software (Thermo) and Prism (GraphPad).

6.2 Results

Brain Metastasis Linked to Cx43 Gap Junction Formation

Lung and breast cancers are the most common sources of brain metastasis(Gavrilovic and Posner 2005). We employed four brain metastatic modelsderived from mammary (MDA231-BrM2, ErbB2-BrM) or lung adenocarcinomas(H2030-BrM3, Kras/p53-BrM), of either human or murine origin (FIG. 6a )(Bos, Zhang et al. 2009, Nguyen, Chiang et al. 2009, Winslow, Dayton etal. 2011, Valiente, Obenauf et al. 2014). When implanted as orthotopictumours or inoculated into the arterial circulation of mice, these cellsform lesions that replicate key histopathologic features of brainmetastasis, including marked astrocytosis (FIG. 1a )(Bos, Zhang et al.2009, Nguyen, Chiang et al. 2009, Valiente, Obenauf et al. 2014). In allthese models, brain metastatic cells produce anti-PA serpins to preventgeneration of lethal plasmin by reactive astrocytes (Valiente, Obenaufet al. 2014). However, co-culture with astrocytes protected cancer cellsfrom chemotherapy and the pro-apoptotic cytokine FasL (FIG. 6b ),congruent with previous in vitro findings (Kim, Kim et al. 2011). Theseresults suggested a possible dual role of astrocytes in brainmetastasis.

Astrocytes interact in a vast gap-junction network (Theis and Giaume2012, Haydon and Nedergaard 2015). Connexin 43 (Cx43) is one of theprincipal gap junction proteins in astrocytes. In our brain metastaticmouse model, we observed Cx43 expression at the interface of cancercells and surrounding astrocytes (FIG. 1b ). Cx43 can mediateinteractions between cancer cells and endothelial cells (Cai, Jiang etal. 1998) and astrocytes (Zhang, Iwakuma et al. 2009) proposed to bepro-metastatic (Pollmann, Shao et al. 2005) or anti-metastatic (Sharma,Abraham et al. 2010). To determine the clinical association of Cx43 withbrain metastasis, we assayed patient tissue samples. In triple-negativebreast cancer and non-small cell lung cancer (NSCLC), we found a higherlevel of Cx43 staining in brain metastases than in primary tumours ornormal tissues (FIG. 1c-d ).

Gap junctions are formed by hexameric connexin hemi-channels. Pairwiseinteractions between hemi-channels on adjacent cells form pores for thetraffic of cytosolic molecules (Bennett and Goodenough 1978, Oshima2014). Not all gap junctions form functional pores (Stoletov, Strnadelet al. 2013), (Sharma, Abraham et al. 2010). However, we observedtime-dependent transfer of calcein from brain metastatic cells toastrocytes, as shown by time-lapse fluorescence microscopy (FIG. 1e ;FIG. 6c ), and from astrocytes to metastatic cells, as shown by flowcytometry (FIG. 6d ).

Brain Metastases Upregulate Protocadherin 7.

Astrocyte calcein transfer occurred more readily with brain metastaticcells than with their parental counterparts (FIG. 1f ). This phenotypewas not fully explained by higher Cx43 expression in the brainmetastatic derivatives (FIG. 1g , FIG. 7a,b ). Moreover, Cx43 expressionin the metastatic cells was lower than, or similar to that inastrocytes, neurons, or brain microvascular endothelial cells (FIG. 1h ,FIG. 7c ). The expression level of other astrocytic connexins (Cx26,Cx30) in brain metastatic cells was similar to that of parental cells(FIG. 7d ). These observations raised the question of how metastaticcells could compete for gap junction formation with resident astrocytes.

Reasoning that cancer cells must use another component besides Cx43 toengage astrocytes, we investigated protocadherin 7 (PCDH7), one of asmall group of genes that are upregulated in brain metastatic cells fromboth breast and lung tumours (Bos, Zhang et al. 2009, Nguyen, Chiang etal. 2009, Valiente, Obenauf et al. 2014). Protocadherins are integralmembrane proteins with seven cadherin repeats that direct cell-cellcontacts by homophilic interaction. PCDH7 (also known ascadherin-related neuronal receptor) is the sole protocadherin expressedpredominantly in the brain (Yoshida, Yoshitomo-Nakagawa et al. 1998,Kim, Chung et al. 2007); its function is unknown. PCDH7 levels werehigher in brain metastatic derivatives than in parental cell lines (FIG.1g , FIG. 7a,b ) or in matched derivatives that are highly metastatic tobone or lung but not brain (FIG. 1i ; refer to FIG. 6a ). The PCDH7level in brain metastatic cells was higher than in astrocytes, neurons,microglia or endothelial cells (FIG. 1h , FIG. 7c ).

In clinical cohorts of triple-negative breast cancer with site ofrelapse annotation, combined expression of PCDH7 and Cx43 in primarytumours was associated with brain metastasis, but not bone or lungmetastasis (FIG. 1j ). Although most NSCLC datasets are not annotatedwith site-specific metastasis information, a large proportion (up to70%) of relapses in these patients include brain metastases (Gaspar,Chansky et al. 2005). Due to the profound morbidity and mortalityassociated with brain metastases (Gaspar, Scott et al. 2000), thesecontribute disproportionately to metastasis-free survival. Indeed, Cx43and PCDH7 expression was associated with decreased metastasis-freesurvival of NSCLC patients in three cohorts (FIG. 1k , FIG. 7e ). Theseresults all support the hypothesis that PCDH7 and Cx43 are relevant inbrain metastasis.

PCDH7 Directs Carcinoma-Astrocyte Gap Junctions.

Brain-metastatic cells depleted of either PCDH7 or Cx43 by means ofshort hairpin RNAs (shRNA) (FIG. 7f,g ) showed reduced capacity for dyetransfer to astrocytes compared to controls (FIG. 2a , FIG. 8a ). Theextent of dye-transfer inhibition after Cx43 depletion was comparable tothat obtained with the pan-connexin inhibitor, carbenoloxone (FIG. 8b ).Given the ability of cadherins to establish homophilic binding betweenmolecules on adjacent cells (Yagi and Takeichi 2000), we hypothesizedthat astrocyte PCDH7 might participate in the formation of gap junctionswith cancer cells. Indeed, PCDH7 depletion in astrocytes (FIG. 8c ) alsoinhibited dye transfer from MDA231-BrM2 cells (FIG. 8d ).

Human brain microvascular endothelilal cells (HBMECs) express much lowerlevels of Cx43 than astrocytes, and have no detectable PCDH7 expression(FIG. 1h , FIG. 7c ). A low level of PCDH7-independent gap junctioncommunication occurred between cancer cells and HBMECs (FIG. 8e ). In acompetition experiment, dye transfer between cancer cell and astrocytewas favored over dye transfer between cancer cell and endothelial cell(FIG. 8f ). Primary microglia cells expressed very low levels of Cx43and PCDH7 and did not accept calcein from cancer cells (FIG. 8g ). Cx43levels in astrocytes and cancer cells remained constant after co-culturewith microglia (FIG. 8h ). Thus, PCDH7 directs cancer cells topreferentially form Cx43 gap junctions with astrocytes.

We employed a split luciferase complementation assay (Luker, Smith etal. 2004) to detect PCDH7 interactions with Cx43 in live cells.Constructs encoding PDCH7 and Cx43 fused to the N-terminal (NLuc) andC-terminal (CLuc) halves of firefly luciferase were expressed inrelevant combinations in non-GFP-luciferase labeled parental cells (FIG.2b ). When NLuc and CLuc come into proximity, luciferase activity isreconstituted. Because Cx43 self-assembles into hexameric semi-channelsin the cell membrane, transduction of cells with Cx43-NLuc and Cx43-CLucvectors served as positive control (FIG. 2b ). We detected specificluciferase activity in cells expressing both Cx43-CLuc and PCDH7-NLuc(FIG. 2b ). The expression level of PCDH7 and Cx43 was higher than theendogenous levels in the parental cells but lower than, or comparable tothe levels in brain metastatic cells (FIG. 9a ). Moreover, co-culturewith astrocytes increased the luciferase signal in the cancer cells(FIG. 9b ) suggesting that astrocyte Cx43 and PCDH7 induce furtherclustering of cancer cell Cx43-CLuc and PCDH7-NLuc. No activity wasdetected when N-cadherin or E-cadherin were fused with NLuc andco-expressed with Cx43-CLuc (FIG. 9c-e ).

Cx43 and PCDH7 Mediate Brain Metastatic Colonization.

shRNA-mediated depletion of either Cx43 or PCDH7 inhibited formation ofbrain metastases by breast cancer and lung cancer cells in xenograft(FIG. 2c-d ) and immunocompetent models (FIG. 10a ). Immunohistologicstaining for GFP in brain sections confirmed this result anddemonstrated a marked reduction in lesion size as a result of Cx43 orPCDH7 depletion (FIG. 10b ). Depletion of Cx43 or PCDH7 did not affectthe formation of lung lesions by MDA231-BrM2 cells after tail veininjection (FIG. 10c ).

Because connexins may mediate cell-cell interactions independently ofchannel function, we employed the Cx43(T154A) mutant that lacks channelfunction but still assembles hemichannels (FIG. 2e ) (Beahm, Oshima etal. 2006). Cx43, either wild type or T154A mutant, was re-expressed inCx43-depleted brain metastatic cancer cells (FIG. 10d ). The mutant Cx43was unable to mediate calcein transfer from astrocyte to MDA231-BrMcells (FIG. 2e ). Wild-type Cx43 rescued brain metastatic activity inCx43-depleted MDA231-BrM and H2030-BrM cells, whereas Cx43(T154A) didnot (FIG. 2f , FIG. 10e ). Together, these observations support a modelin which PCDH7 directly and specifically interacts with Cx43 toselectively promote functional gap junction formation between cancercells and astrocytes (FIG. 2g ).

To define the stage at which PCDH7 and Cx43 contribute to the formationof brain metastases, we performed short-term metastasis assays withMDA231-BrM2 cells. In this model, extravasation across the BBB iscomplete 7 days post-inoculation, vascular cooption and overt outgrowthoccur by day 14 (Valiente, Obenauf et al. 2014). Cx43 or PCDH7 depletionin the cancer cells did not significantly diminish the number of GFP+cancer cells in the brain parenchyma 7 days after inoculation (FIG. 11a). Fourteen days after inoculation, micrometastases resulting from Cx43or PCDH7 depleted cells showed decreased proliferation, as determined byKi67 staining (FIG. 11b ). Apoptosis of brain metastatic cells wasdetermined in the ex-vivo brain slice assay (Valiente, Obenauf et al.2014). With this approach, we found increased caspase 3 staining in Cx43or PCDH7-depleted cells, consistent with increased apoptosis. (FIG. 11c). Of note, the Cx43-depleted or PCDH7-depleted cells were still able toclosely interact with capillaries (FIG. 11d ). Thus, cancercell-astrocyte gap junctions support brain metastasis development afterinitial extravasation and vascular cooption.

Cancer Cells Gap Junctions Trigger Astrocyte Cytokine Release.

To determine the mechanism behind this Cx43-mediated brain metastaticgrowth, we employed translating ribosome affinity purification (TRAP)(Heiman, Schaefer et al. 2008) to assay cancer cell gene expression inmixed co-cultures (FIG. 12a ). We expressed the eGFP-tagged L10aribosomal subunit in MDA231-BrM2 cells with either basal or reduced Cx43expression. After cancer cell co-culture with astrocytes for 24 h, eGFPimmunoprecipitation and polysome-associated mRNA harvest from cancercells was followed by global transcriptome sequencing (TRAP-RNAseq)(FIG. 12 b.c). Gene signature analysis revealed that the interferon(IFN) and NF-κB pathways were the most activated pathways in brainmetastatic cells after co-culture with astrocytes, and these effectsrequired Cx43 (FIG. 3a ). Other upregulated pathways included Her2/AKTand TGFβ. Conditioned media from astrocyte-MDA231-BrM2 co-cultures wassufficient to activate the IFN and NF-κB signaling in the cancer cells,as determined by increased phosphorylation of STAT1 and NF-κB p65 (FIG.3b , FIG. 12d ). This effect was not observed with conditioned mediafrom astrocyte co-cultures with Cx43-depleted or Cx43(T154A)reconstituted cancer cells (FIG. 3c ).

Analysis of conditioned media generated in MDA231-BrM2-astrocyteco-cultures (FIG. 3d ) demonstrated accumulation of type I interferon,IFNα, and TNFα in a gap-junction dependent manner (FIG. 3e , FIG. 13a-b); no type II interferon, IFNγ, was detected (data not shown).MDA231-BrM2, either alone or co-cultured with astrocytes, did notexpress these cytokines as detected by TRAP-RNAseq (data not shown).Upregulation of INFα and TNFα mRNA was detected in the astrocytesreisolated after the co-culture (FIG. 3f ). These results suggested thatthe heterocellular gap junction communication elicited production ofIFNα and TNFα in astrocytes, triggering STAT1 and NF-κB pathwayactivation in the cancer cells.

Addition of IFNα and TNFα inhibited the apoptotic response of brainmetastatic cancer cells to cytotoxic chemotherapy in vitro (FIG. 3g ,FIG. 13c ). To assess the functional importance of these pathways inbrain metastasis, we knocked down STAT1 by shRNAs (FIG. 3h , FIG. 13d )or inhibited NF-κB by overexpression of IκBα super suppressor (SR-IκBα)(Boehm, Zhao et al. 2007) (FIG. 3i ) in brain metastatic cells. Wheninoculated into mice, these cells produced smaller brain metastases thancontrol counterparts (FIG. 3j , FIG. 13e ), suggesting that STAT1 andNF-κB activators provide a survival advantage for metastatic cells inthe brain.

Cancer Cell Gap Junctions Activate the Cytosolic dsDNA Response inAstrocytes.

Whereas IFNα and TNFα may be individually induced by diverse inputs, thejoint upregulation of both cytokines was reminiscent of a cellularresponse to cytosolic double stranded DNA (dsDNA) (Cai, Chiu et al.2014). Cytosolic dsDNA triggers the cGAS-STING pathway, in which cyclicGMP-AMP synthase (cGAS) senses cytosolic dsDNA and synthesizes thesecond messenger 2′3′-cyclic GMP-AMP (cGAMP). cGAMP binding to STINGtriggers phosphorylation and activation of TBK1 and IRF3, nuclearaccumulation of IRF3, and transcriptional activation of IRF3 targetgenes IFNA and TNFA (Wu, Sun et al. 2013). This pathway represents anancient anti-viral innate immune response (Cai, Chiu et al. 2014).

Co-incubation of MDA231-BrM2 cells and astrocytes triggeredphosphorylation of TBK1 and IRF3 in a Cx43-dependent manner (FIG. 4a ,FIG. 14a ). Nuclear accumulation of IRF3 occurred only in the astrocytesin co-cultures, and not in astrocytes or cancer cells cultured alone(FIG. 4b ). Using LC-MS/MS, we detected cGAMP in MDA231-BrM2 cells butnot in astrocytes cultured alone (FIG. 4c-d , FIG. 14b ). Co-culture ofa fixed number of MDA231-BrM2 cells with astrocytes led to aCx43-dependent increase in the levels of cGAMP (FIG. 4c-d ). Usingstress conditions that release mitochondrial dsDNA into the cytosol, weconfirmed that astrocytes are competent to produce cGAMP in response tocytosolic dsDNA (Rongvaux, Jackson et al. 2014).

Subcellular fractionation demonstrated that these brain metastatic cellsand other human cancer cell lines contain cytosolic dsDNA whereasastrocytes and other non-neoplastic human cells do not (FIG. 4e , FIG.14c,d ). By immunofluorescence, we detected cytosolic dsDNA in brainmetastatic cancer cells (FIG. 4f , FIG. 9e ), but not in astrocytes(FIG. 14f ). To determine if cancer cell DNA passes to astrocytesthrough Cx43 gap junctions, we labeled cancer cell DNA with5-ethynyl-2′-deoxyuridine (EdU), co-cultured the cells with astrocytesand analyzed the distribution of labeled DNA by microscopy (FIG. 4g ,FIG. 14g ) or flow cytometry (FIG. 4h ). Both methods demonstratedtransfer of DNA from the cancer cell to the astrocyte in aCx43-dependent manner.

Taken together, these results support a model in which brain metastaticcancer cells contain cytosolic dsDNA and cGAMP, and employ PCDH7 toengage astrocytes in Cx43-based gap junctions. The gap junctions allowpassage of cytosolic dsDNA (and cGAMP) from cancer cells into astrocytesto trigger the generation of additional cGAMP, TBK1 and IRF3 activation,and production of IFNα and TNFα. Acting as paracrine factors, thesecytokines activate STAT1 and NF-κB signaling in the cancer cells, whichsupport the growth and survival of the cancer cells in the face ofmicroenvironmental and chemotherapeutic stresses (FIG. 4i ).

Pharmacologic Inhibition of Gap Junction Activity.

The evidence that genetic inhibition of gap junction componentsdecreased brain metastatic outgrowth provided a rationale for testingpharmacologic suppressors of gap junction activity against brainmetastasis. To this end, we selected two orally bioavailable compoundsfor pre-clinical trials. In addition to anti-inflammatory activity,meclofenamate inhibits Cx43 gap junction gating (Harks, de Roos et al.2001), inhibits epileptogenesis in animal models (Jin, Dai et al. 2013),passes the BBB after systemic administration (Harks, de Roos et al.2001), is well tolerated systemically (Holmes 1966) and is currently anFDA-approved NSAID. Tonabersat is an benzopyran derivative that binds toa unique stereoselective binding site in astrocytes (Herdon, Jerman etal. 1997, Chan, Evans et al. 1999), inhibits gap-junction-mediatedpathophysiological processes including cortical spreading depression(Read, Smith et al. 2000) and trigeminal ganglion neuronal-satellitecell signaling in animal models (Damodaram, Thalakoti et al. 2009), andwas systemically well-tolerated and safe in patients with migraine(Dahlof, Hauge et al. 2009).

Both Tonabersat and meclofenamate inhibited dye transfer from astrocytesto cancer cells as measured by flow cytometry (FIG. 5a ), and therelease of IFNα and TNFα in co-cultures of these cells (FIG. 5b ),recapitulating the phenotype seen in knockdown of Cx43 or PCDH7. Micewere treated with either vehicle or with these compounds from day 1following arterial inoculation of MDA231-BrM2 cells or H2030-BrM3 cellsin immunodeficient mice, or KRas/p53-393N1 cells in immunocompetent mice(FIG. 5c , FIG. 15a,b ). Both drugs prevented the emergence of brainmetastases, consistent with our evidence that gap junction activity isrelevant for metastatic outgrowth. However, this treatment did notrestrict growth of MDA231-BrM2 cells as lung metastatic lesions or asorthotopic tumours (FIG. 15c, d ).

Gap Junction Directed Therapy.

To test the effect of Cx43 or PCDH7 depletion in established metastases,we transduced MDA231-BrM2 cells with Tet-inducible shRNA expressionvectors (FIG. 5e ). A red fluorescence protein (RFP) under the controlof the same promoter provided a marker of hairpin expression in vivo(FIG. 10e ). Cells transduced with inducible Cx43 or PCDH7 shRNA vectorsshowed doxycycline-dependent depletion of Cx43 or PCDH7, respectively(FIG. 15f ). These cells were injected intracardially and allowed toform brain metastases for 14 days. At this stage, brain lesions areapparent by BLI in all mice (FIG. 15g ); the aggressive lesions engulfthe microvasculature (FIG. 5d ) and will result in death of the animalsin 2-3 weeks (Bos, Zhang et al. 2009, Valiente, Obenauf et al. 2014).Doxycycline administration starting on day 14 resulted in reduced brainmetastatic burden three weeks later, compared to controls (FIG. 5f,g ).

Brain metastases are distinguished by pronounced resistance tochemotherapy (Zhang, Price et al. 1992, Deeken and Loscher 2007).Carboplatin crosses the BBB (Pitz, Desai et al. 2011), with modestimprovement in overall survival in patients with brain metastases frombreast (Lim and Lin 2014) or lung cancer (Taimur and Edelman 2003).Carboplatin alone (50 mg/kg/5 days) starting on day 14 inhibited brainmetastasis to a similar extent as depletion of Cx43 or PCDH7 (FIG. 5f,g); combination carboplatin and doxycycline reduced the metastatic burdenfurther (FIG. 5f,g ). Therefore, we assessed the effectiveness ofcombination gap junction modulatory therapy with chemotherapy (FIG. 5h). Treatment with carboplatin alone minimally inhibited brain metastasisgrowth (FIG. 5i ). Either Tonabersat (10 mg/kg) or meclofenamate (20mg/kg) as single agents (FIG. 5i ) significantly inhibited progressionof metastatic lesions at the 35-day end point. The combination ofcarboplatin with either Tonabersat or meclofenamate profoundly inhibitedbrain metastasis (FIG. 5i ).

6.3 Discussion

The brain represents a unique and formidable metastatic target, withastrocytes a predominant feature of the microenvironment. We presentevidence that cancer cells employ PCDH7 to selectively engage astrocytesin vital Cx43 gap junctions. Cadherin family members are importantmediators of cell-cell communication in development and tissuehomeostasis (Yagi and Takeichi 2000), particularly in the nervous system(Hirano, Suzuki et al. 2003). It is remarkable that brain metastaticcells adopt a particular member of this family whose normal expressionis largely restricted to the brain (Yoshida, Yoshitomo-Nakagawa et al.1998). PCDH7 therefore joins ST6GALNAC5 (Bos, Zhang et al. 2009), andneuroserpin (Valiente, Obenauf et al. 2014) as brain-restrictedcomponents that brain metastatic cells from breast and lung carcinomasselectively express to colonize the brain.

PCDH7 and Cx43 contribute to brain metastatic colonization andchemoresistance. Functional Cx43-based gap junctions between cancercells and astrocytes allow cancer cells to disseminate cytosolic dsDNAto the astrocyte network. This activates the astrocytic cGAS-STINGpathway, culminating in release of cytokines including IFNα and TNFα.These cytokines provide a growth advantage for brain metastatic cells byprotecting against physiologic and chemotherapeutic stressors. Otherupregulated pathways include Her2/AKT and TGFβ. Our results thereforeprovide in vivo evidence and mechanistic underpinnings for a previouslyobserved chemoprotective effect of astrocytes on cancer cells in vitro(Kim, Kim et al. 2011). The present evidence together with previous worksuggests that cancer cells protect themselves from astrocytic attack intwo ways, first, through production of serpin inhibitors of cytotoxicplasmin generation, and second, by engaging astrocytes through gapjunctions and appropriating the dsDNA response.

Cytosolic dsDNA was first defined as an activator of innate immunityagainst viral infection (Stetson and Medzhitov 2006). In cancer cells,there are a number of possible sources of dsDNA including genomicinstability, mitochondrial stress, and exposure to DNA-damaging agents.DNA-triggered innate immune responses and, specifically, cGAMP, can passto other cells through gap junctions (Patel, King et al. 2009, Ablasser,Schmid-Burgk et al. 2013). Fitting with these observations, we find thatmalignant cells, including brain metastatic derivatives, contain highlevels of cytosolic dsDNA and cGAMP compared with astrocytes and otherstromal cells. Importantly, in brain metastasis the dsDNA responseemerges from intrinsic cytosolic dsDNA in the cancer cells, isCx43-dependent, and involves host tissue astrocytes, thus representingan unprecedented pro-metastatic process.

Brain metastases are a major contributor to cancer patient morbidity andmortality, with few therapeutic options available. Early steps in thebrain metastatic cascade, including cancer cell dissemination andextravasation through the BBB, have not been amenable to therapy (Maher,Mietz et al. 2009, Eichler, Chung et al. 2011). However, cancer celldependency on Cx43/PCDH7 gap junctions for survival and outgrowth ofmetastatic lesions suggests a therapeutic opportunity. Our pre-clinicalresults using combinations of chemotherapy and gap junction modulatorsprovide proof-of-principle for the therapeutic potential of theseinterventions against brain metastasis.

TABLE 1 Target Sequences of shRNAs (SEQ ID NOS: 1-14, top to bottom)PLKO.1 lenivirus vectors-human genes Name of sh Catalog number SequenceCx43 sh1 TRCN0000059773 GCCCAAACTGATGGTGTCAA T Cx43 sh2 TRCN0000059775GCGACAGAAACAATTCTTCTT PCDH7 sh1 TRCN0000055744 GCAGGAGACAACATTTCAATPCDH7 sh2 TRCN0000291663 GCTGGCATTATGACGGTGAT T STAT1 sh1 TRCN0000280021CTGGAAGATTTACAAGATGAA STAT1 sh2 TRCN0000004265 CCCTGAAGTATCTGTATCCAATRIPZ inducible lenivirus vectors-human genes Name of sh Catalog numberSequence Cx43 sh1 V3THS_411733 TAAGGACAATCCTCTGTCT Cx43 sh2 V3THS_411729TGAGTGGAATCTTGATGCT PCDH7 sh1 V3THS_338930 GAATCAACACTGCCATCCG PCDH7 sh2V3THS_152694 TTAAGATGATTAGAATCAC GIPZ lenivirus vectors-mouse genesName of sh Catalog number Sequence Cx43 sh1 V3LHS_411730TGAGTACCACCTCCACCGG PCDH7 sh1 V3LMM_510718 TAACTTTAAACTCATACCT PCDH7 sh2V2LMM_11270 TAAACTTAGGGTCGTTGTC Control sh Name of sh Ctrl sh SHC016CCGGGCGCGATAGCGCTAAT AATTTCTC

TABLE 2 Antibodies Catalog Antibody against Company number Westernblotting antibodies Cx43 Cell Signaling 3512 PCDH7 Sigma-AldrichHPA011866 α-tubulin Sigma-Aldrich T6074 E-cadherin Cell Signaling 3195N-cadherin Sigma-Aldrich C3865 Phospho-STAT1 Cell Signaling 9167 STAT1Cell Signaling 9172 Phospho-NF-κBp65 Cell Signaling 3033 NF-κB p65 CellSignaling 8242 Phospho-TBK1 Cell Signaling 5483 TBK1 Cell Signaling 3013Phospho-IRF3 Cell Signaling 4947 IRF3 Cell Signaling 11904 IκBα CellSignaling 4812 Immunochemical staining antibodies Cx43 Cell Signaling3512 GFP Aves Labs GFP-1020 Ki67 Vector VP-K451 Laboratories GFAP DakoZ0334 GFAP EMD Millipore MAB360 CollagenIV EMD Millipore AB756P IRF3Cell Signaling 9172 dsDNA EMD Millipore MAB1293 CoxIV Cell Signaling4850

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Various references are cited herein, the contents of which are herebyincorporated by reference in their entireties. Various nucleic acid andamino acid sequence accession numbers are cited herein, and the completesequences referenced by those accession numbers are hereby incorporatedby reference in their entireties.

What is claimed is:
 1. A method for treating a cancer that hasmetastasized to the brain in a subject, comprising: administering to thesubject tonabersat.
 2. The method of claim 1, wherein the tonabersat isadministered in an amount of about 10 mg/kg.
 3. The method of claim 1,wherein the tonabersat is administered in an amount of between about 0.8mg/kg and about 1.2 mg/kg.
 4. The method of claim 1, wherein the canceris a breast cancer or a lung cancer.
 5. The method of claim 1, furthercomprising administering to the subject an anti-cancer agent
 6. Themethod of claim 5, wherein the anti-cancer agent is carboplatin.
 7. Themethod of claim 6, wherein the carboplatin is administered in an amountof between 4 mg/kg and about 6 mg/kg.
 8. A method for preventing and/orinhibiting the metastasis of a cancer to the brain in a subject,comprising: administering to the subject tonabersat.
 9. The method ofclaim 8, wherein the tonabersat is administered in an amount of about 10mg/kg.
 10. The method of claim 8, wherein the tonabersat is administeredin an amount of between about 0.8 mg/kg and about 1.2 mg/kg.
 11. Themethod of claim 8, wherein the cancer is a breast cancer or a lungcancer.
 12. The method of claim 8, further comprising administering tothe subject an anti-cancer agent
 13. The method of claim 12, wherein theanti-cancer agent is carboplatin.
 14. The method of claim 13, whereinthe carboplatin is administered in an amount of between 4 mg/kg andabout 6 mg/kg.
 15. A method for reducing the risk of the metastasis of acancer to the brain in a subject, comprising: administering to thesubject tonabersat.
 16. The method of claim 15, wherein tonabersat isadministered in an amount of about 10 mg/kg.
 17. The method of claim 15,wherein the tonabersat is administered in an amount of between about 0.8mg/kg and about 1.2 mg/kg.
 18. The method of claim 15, wherein thecancer is a breast cancer or a lung cancer.
 19. The method of claim 15,further comprising administering to the subject an anti-cancer agent 20.The method of claim 19, wherein the anti-cancer agent is carboplatin.